3 11 Presumed photoreceptive cilia in a ctenophore By G. A. HORRIDGE (From the Gatty Marine Laboratory and Department of Natural History, the University, St. Andrews, Fife) With 6 plates (figs, i to 6) Summary Four groups of lamellate bodies are symmetrically arranged inter-radially in the floor of the apical organ. Each is composed of many streamed-out membranes of a group of about a dozen cilia, within an invagination of a cell. The rings of ciliary fibrils, of the 9 + 0 pattern, become disarrayed not far from the base. Their dense membranes are lined with granules so that the successive lamellae resemble those of the modified cilia of vertebrate eyes, although flattened in a different plane. On this basis, of ciliary origin, and their resemblance to photoreceptors in fine detail, these structures are interpreted as photoreceptors. Introduction IT is commonly accepted that ctenophores have no special light-sensitive organ. But in his monograph on the ctenophores of Naples, Chun (1880) shows 4 crescent-shaped groups of round bodies, each of which is about the size of a nucleus, symmetrically placed about the floor of the apical organ. Chun finds these in all ctenophores and suggests (p. 166) that they are primitive photoreceptor organs, but hardly to be called an eye. Responses of these animals to light are also supposed to be indefinite (Hyman, 1940). No reports of immediate or rapid responses to illumination or darkening have come to hand, and tests show none in Pleurobrachia, although it is possible that ctenophores migrate vertically in the plankton in relation to diurnal light changes. However, upon close examination at the electron-microscope level, the organs mentioned by Chun turn out to have a structure which is remarkably similar to that of photoreceptors in the eyes of vertebrates (Brown, Gibbons, and Wald, 1963) and in the distal retina of the clam Pecten (Miller, 1958), in both of which, in different ways, the folded membranes of the photoreceptors are derived from the membranes of cilia. Recently Eakin (1963) has found that the photoreceptors of starfish, coelenterates, and Sagitta are also complicated elaborations of cilia. The justification for regarding as photoreceptors the structures to be described in ctenophores rests only on this anatomical similarity. Direct electrophysiological micro-methods would be necessary to prove the case, because otherwise it is difficult to experiment with only one group of the diversely differentiated cells of the ctenophore apical organs. [Quart. J. micr. Sci., Vol. 105, pt. 3, pp. 311-317, 1964.]
312 Horridge Ctenophore photoreceptor Methods Pleurobrachia pilots, collected from the plankton of St. Andrews bay, was fixed in 2 % osmic acid mixed with an equal volume of sea-water of ph 7-2 to 7-4. As in coelenterates, the tissue is very watery and it is difficult to achieve good fixation of a variety of components and tissues at the same time, no matter what combinations of dilution and added sucrose are tried. Apical organs were embedded in araldite, and sectioned and stained with lead acetate, according to the alcohol-ether method described in a previous paper (Horridge and Mackay, 1964). The correct region of the apical organ was identified by examination of 1 /x sections stained in aqueous toluidine blue (fig. 1, A, B). Results As seen with the light microscope in horizontal or vertical sections of the apical organ there are 4 groups of round basiphilic objects in the rounded angles at the base of the side wall (fig. 1, A). Each group consists of up to 20 lamellated objects, each of 4 to 8 ju. in diameter. Here and there an individual of the group is associated with a space or vacuole. Each of these objects lies separate and distinct from its neighbours within the thickness of the ectodermal epithelium of the apical organ. The epithelium consists of tightly packed columnar cells up to 100 fi tall, which are of several types distinguished by their contents. These oval dark objects are quite distinct from the developing grains of the statolith, which lie in a different region of the wall of the apical organ. Under the electron microscope each of these objects is resolved as a lamellate body (figs. 2, 3) of up to 50 coiled membranes which are thicker than normal plasma membranes and which, when fixed and stained as described, become electron-dense (figs. 4, 5). The membranes are those of cilia which have extended sideways, on one side or on both sides of their axis, and the membranes have coiled around themselves and round each other within the ambit of a cavity. This cavity originates as an invagination of the plasma membrane of the cell where its distal end meets the sea-water in the cavity of the apical organ. Apart from the directions of the fibrils of the cilia, the structure of one of these lamellate bodies appears fairly similar no matter FIG. I (plate), A, horizontal (equatorial plane) section through the apical organ of Pleurobrachia, just grazing the underside of the statolith, which is visible in the centre. This section is transverse to the main axis of symmetry of the animal. The top 4 groups of lamellate bodies are just beginning to come into view at this level. B, a section, deeper in the same series, parallel to the above but through the ectodermal floor of the apical organ, showing the 2 groups of lamellate bodies on one side. This is about the maximum number found in any one section. c, a typical cilium of the 9 + 0 type, of a cell of the side wall of the apical organ, in the region above the lamellate bodies, which are generated from cilia of this type. D, a cilium of the 9 + 2 type, which is abundant in the apical organ. b, otolith balancers; c, cilia at the beginning of the ciliated groove; end, endoderm; o, lamellate bodies; J, sagittal plane (Hyman, 1940); st, statolith.
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Horridge Ctenophore photoreceptor 313 what plane it is sectioned in, showing that they are spherical and not cylindrical in general form. The exact pattern of the membrane differs in each lamellate body. However, the same fundamental relationship between the membranes of the cilia and the plasma membrane of the vacuole occurs in all. A group of between 6 and 12 cilia of the 9+0 pattern (fig. 1, c) spring from the wall of the invagination in the cell on its side towards the cavity of the apical organ. As can be followed more clearly in lamellate bodies which are interpreted as growing ones (see below), the membranes of the cilia spread progressively out sideways until they have circled round the inside of the vacuole (figs. 3, A, B; 6, c). This process of membrane extension happens on one side (figs. 3, B; 4, A) or on both sides (figs. 5, c; 6, c) of the axis of the cilium. The lamellate body is built up of successive turns as the membranes extend in the spaces available to them. The overlapping of adjacent layers becomes extremely complicated because the process happens by growth of structures, topologically the shape of folded pieces of paper, which come to fit into layers within a sphere. Frequently the plane of a section includes some cilia which are seen in longitudinal section and others which are cut more transversely (fig. 5, B). Because they are the extensions of the side wall of a tube, the membranes are paired and the 2 membranes of a pair meet to form a loop either in the centre of the lamellate body or, more commonly, against the plasma membrane of the invagination in which the lamellate body lies (fig. 4, A, B, C, in different patterns). Finger-like protrusions from the edges of the folded membranes become sectioned and appear as totally enclosed vesicles with walls which are not seen as being continuous with the folded ends of adjacent lamellae (figs. 3, B; 6, B). Brown, Gibbons, and Wald (1963) consider that similar vesicles at the ends of folded lamellae in vertebrate rods and cones are not artefacts: it is necessary to say this in view of criticisms of fixation by osmic acid (Rosenbluth, 1963). The membranes lie in pairs and, in many examples, the spaces between them can be traced back to the inside and outside of the cilium which gave rise to them: the outside surface is the smooth one (fig. 4, A). The inside surface of each membrane is rough and covered with granules which add to the thickness of the membrane (fig. 4, A). In situation and appearance the granules closely resemble those thought to contain the photoreceptor pigments in vertebrate eyes, as for example discussed by Brown, Gibbons, and Wald (1963) in relation to the lamellar micelles in the cones of the amphibian Necturus. In the vertebrate photoreceptors the lamellar membranes are invaginations of the cilium membrane and have the granules on the outside Fie. z (plate). A group of lamellate bodies in the side wall of the apical organ, cut in a sagittal plane (parallel to the principal axis at right angles to the line between the tentacles). The cells containing the lamellate bodies have a distinct complement of vesicles and neurptubules. The lamellate body which is surrounded by loose membranes (centre right) is one which is degenerating, and all stages to final dissolution have been found. The mesogloeal side is towards the bottom.
314 Horridge Ctenophore photoreceptor surfaces of a fold, whereas here they are extensions and have the granules on the inside. The lamellate bodies of the ctenophore Callianira were reported as being brownish-red by Kolliker (1853). The regularity of the spacing between the membranes is outstanding in some isolated regions of lamellate bodies but in most places the regular spacing is only between the outsides of 2 cilia. The distance, where it is regular, between outer ciliary surfaces is consistently 10 to 12 m/x: whereas between inner surfaces it varies between 15 m/x and 100 m/a. This invites the interpretation that the constant separation between cilia is maintained by the regular arrangement of a solid packing which is, however, not seen. Other cilia in ctenophores, such as those of the comb-plates and the balancers, are fused closely together in groups with a regular spacing, again suggesting an adhesion of the outer surfaces. The cilia which give rise to the lamellate bodies have the 9+0 pattern typical of sensory cilia (Barnes, 1961) including the vertebrate rods and cones, and the photoreceptors of Pecten. In the apical organ there are several other types of cilia, some of the 9+2 pattern with a specialized root structure which lies at right angles to the cilium shaft, others with the 9+0 pattern. The cilia of the lamellate body differ from all these in having the granules which suggest photopigments. The basal structure is different from that of any other cilium which I have seen described. The basal body of each cilium contains the 9 triple fibrils which are characteristic of a centriole (fig. 5, A). Certainly 1 and probably 2 of the fibrils of the basal body triplets are continuous with the paired fibrils of the cilium (figs. 4, B; 5, B; 6, B). The basal body is not at an angle to the cilium shaft and it lies just inside the meeting of the cilium membrane with the plasma membrane. A structure resembling the spokes of a cartwheel projects outwards from the sides of the basal body (fig. 5, A, left). There is no definite root structure, in contrast to ctenophore comb-plate cilia, which have branched roots more or less parallel to their axis, and apical organ balancer cilia, each of which has a striated flattened root at right angles to the shaft. Occasionally a few fibres lie in the cytoplasm around the basal body (fig. 4, B). Commonly there are small vesicles which can lie within the hollow of the cilium shaft (fig. 5, B). The appearance of the basal body in longitudinal sections suggests that a fine amorphous substance lies in the centre of the shaft both in growing and in mature examples. This varies slightly in position in relation to the base and can form cross bands (fig. 4, B). Distally the filaments of the cilium become disarranged (figs. 3, B; 4, A; 6, c) and eventually they peter out. The FIG. 3 (plate). Details of a lamellate body which is interpreted as being in process of development. The invaginated cavity is not yet filled and many of the 9 + 0 type cilia still have recognizable round outlines. B is a high power picture of the relevant region of the top portion of A. ' a, amorphous bodies, which are commonly present; I, longitudinal section of basal body; t, transverse section of basal body; va, vacuole.
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Horridge Ctenophore photoreceptor 315 paired 'hooks' typical of the filaments of normal cilia (fig. 1, D) are not found in the 9+0 pattern (fig. 1, c). The dissociation of the filaments shows that they are less closely bound together laterally than are those of the normal 9-)-2 type characteristic of unspecialized ctenophore cells, apical organ balancers, ciliated grooves, and comb-plates. The size of the ring of 9 is the same in both types of cilia. Typical cilia of the side wall of the apical organ are shown in fig. 1, c (9+0 pattern) and fig. 1, D (9+2 pattern). Other features of the cells which produce the lamellate bodies are those of sensory nerve-cells elsewhere in coelenterates and ctenophores. The cells are elongated and their bases terminate among other elongated cells and fibres at the base of the epithelium where it adjoins the mesogloea. Occasionally there are typical asymmetrical synapses in this region, with vesicles only in the cytoplasm which can be traced back in a cell containing a lamellate body or to a ciliated cell of the wall of the apical organ. The cytoplasm around the lamellate body contains a great number of vesicles and some neurotubules (canaliculi) which resemble those in nerve-cells elsewhere in the animal. Neighbouring cells, without lamellate bodies, contain different types of vesicles. Unlike other cells, those which bear the lamellate bodies are not ciliated where they meet the cavity of the apical organ; this is to be expected because their basal bodies and cilia lie deep below in the invagination. Instead there is sometimes a concentrated region of orientated endoplasmic reticulum, in which the membranes lie at right angles to the cell surface and appear as if they can open to the sea-water. In this region, on other cells, there are secretory cilia, which are readily identified by their bulky contents and swollen ends. Evidence for a continual production and disintegration of lamellate bodies conies from their size distribution, from the occurrence of degenerate-looking examples, and from the fact that all stages of their formation occur in mediumsized sexually mature animals. The sequence can be picked out from cells which are always situated in a definite pattern. The first recognizable stage is of several cilia of the 9+0 type growing inwards from the surface of the epithelium into a small space which is an invagination of the distal plasma membrane. In every case examined there have been relatively large irregular granular particles lying within the invagination, and the extensions of the cilia membranes sometimes grow around these (fig. 3, A, but best seen in fig. 6, c). FIG. 4 (plate). Membranes of the lamellate body with intercalated ciliaryfilaments,vesicles, and the granular lining of the membrane. The folded ends of the paired membranes meet the plasma membrane which originally surrounded the invaginated space. Note the constancy of separation between the outer, but not the inner surfaces of the ciliary membranes. A, transverse section of cilia. B, longitudinal section of cilia. C, regular membrane folds arrive at the plasma membrane of the vacuole. b, transverse bars in the cilium shaft; cm, paired cell membrane of adjoining cells; d, double membrane formed by the wall of the cilium and the folded end of another cilium. lamella; /, fibrils of a cilium; pm, plasma membrane of the wall of the invagination which holds the lamellate body; v, vesicles within ciliary lamellae.
316 Horridge Ctenophore photoreceptor Near its base, the stem of each cilium usually persists as a round pillar, while the lateral extension of the membrane starts further along it, as can be seen in a series (fig. 5). The largest and best-developed lamellate bodies lie further away from the surface of the epithelium, and degenerate lamellate bodies are always situated towards the mesogloeal side of the group (fig. 2). Discussion Although photoreceptors present a vast array of anatomical diversity throughout the animal kingdom, 2 features which many have in common relate to their fundamental mechanism. There is a multiplication of the area of a membrane, which may be loaded with granules and, in a few investigated examples, is known to be associated with the photoreceptor mechanism. There are at least 3 ways in which the convoluted membrane arises, (a) by successive folding of a membrane, which may be associated with a cilium as vertebrates, (b) by growth of a mass of crowded villi which, pressed together, form a rhabdom, and (c) by the coiling of a membrane to form a lamellate body. Arthropod rhabdoms have not so far been related to cilia, but in the arrow-worm Sagitta a rhabdom stands upon a projection which is a modified sensory cilium (Eakin, personal communication). In developing eyes of the clam Pecten, whirled lamellate bodies are formed from sensory cilia (Miller, 1958), although the relationship between the cilia membranes and the plasma membrane is not clearly shown. Identification of ctenophore lamellate bodies as photoreceptors depends on a combination of morphological features. In a mixed tissue, below the organ level of organization, as here in coelenterates and ctenophores, there are usually no other criteria available for the recognition of cell types; similar difficulties have been encountered in the recognition of nerve-cells in ctenophores. The wide distribution of cilia of the few known examples of the 9+0 pattern, always in situations where a sensory function is known or suspected, is reviewed by Barnes (1961). The present example from ctenophores has a basal body formed from a centriole 'in line' and does not fit the classification by Barnes, who suggests that 9+0 photoreceptor cilia, as in rods and cones, are likely to have a basal body together with an additional centriole. Also relevant to Barnes's discussion is the fact that here and elsewhere in ctenophores there are many cilia per cell, and none has an additional centriole. Onion bodies which are not known to be associated with cilia, and which are not suspected of being photoreceptor organelles, occur commonly FIG. S (plate). Details of bases of cilia of a fully developed lamellate body. The 3 sections, A, B, and C are from the same series, showing regions a few microns apart. A, section through the basal bodies. Note the radiating structures spread out from the fibrils, seen especially in the one on the left. B, 2 stems just above the basal bodies and a cilium in longitudinal section at right angles to the others. No flattened membranes are continuous with the cilium membranes in this section, v, vesicles within cilia. c, a few microns towards the mesogloeal end of the cell, the membranes become continuous with those of the cilia.
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Horridge Ctenophore photoreceptor 317 throughout the animal kingdom, especially in nerve-cells. An onion body is an organelle of concentric membranes of 100 m//. to 5-0/^ in diameter, which appears to lie wholly within the cytoplasm. Where closely parallel membranes are curved in a spherical shape the distinction between spiral and concentric layers depends on the plane of the section. It is necessary to distinguish between lamellate bodies formed by the plasma membrane and onion bodies formed by intracellular membranes. Transport of material down the axes of the cilia presumably occurs during the process of extension of the ciliary membranes, and especially during the subsequent laying down of the granular substance which lines the extended membrane. Transport down the visual cilia perhaps occurs also in the narrow region in the rod and cone cells of vertebrates. There is nothing in the structure of the basal body to suggest mechanisms which could move material along the shaft. Related structures in other cilia may be relevant here. In the apical organ of Pleurobrachia there are cilia with swollen ends which contain numerous small vesicles. The statolith in ctenophores stands upon 4 groups of cilia, called balancers, although its constituent particles are synthesized in the cells below. On account of the difficulty of sectioning the hard otolith, I have not been able to determine whether these cilia distended with vesicles are concerned in the formation of the otolith, although such a relation is likely. Even comb-plate cilia sometimes contain vesicles near their basal ends. It is evident that in the formation of otoliths on sensory cilia, and in the related topic of the transport of energy-rich material which maintains ciliary activity, much remains to be discovered. In ctenophore comb-plates the mitochondria are not closely associated with ciliary rootlets as they are in Amphioxus (Olsson, 1962). Possibly the resemblance between neurotubules (canaliculi) and ciliary fibrils is relevant, in that both may in some way act as lines along which streaming occurs. Ctenophores certainly present many opportunities for analysis of processes in cilia. References Barnes, B. G., 1961. J. Ult. Res., 5, 453- Brown, P. K., Gibbons, I. R., and Wald, G., 1963. J. Cell Biol., 19, 79. Chun, C, 1880. Die Ctenophorendes GolfesvonNeapelundderangrenzendenMeeres-Abschnitte. Leipzig (Engelmann). Eakin, R. M., 1963. In General physiology of cell specialization. Ed. Mazia, D., and Tyler, A. London (McGraw-Hill). Horridge, G. A., and Mackay, B., 1964. Quart. J. micr. Sci. (In the press.) Hyman, L., 1940. The invertebrates Protozoa through Ctenophora. London (McGraw-Hill). Kolliker, A., 1853. Zeit. wiss. Zool., 4, 315. Miller, W. H., 1958. J. biophys. biochem. Cytol., 4, 117. Olsson, R., 1962. J. Cell Biol., 15, 596. Rosenbluth, J., 1963. Ibid., 16, 143. FIG. 6 (plate), A, the peripheral end of a typical cell containing a lamellate body, reaching to the sea-water in the cavity of the apical organ. B, longitudinal section through the base of a cilium showing the close similarity between neurotubules of the cytoplasm and cilia fibrils. C, a group of cilia in an immature lamellate body, showing the initiation of the lamellae. a, amorphous body, round which cilia sometimes wrap; m, mitochondrion; n, neurotubules (canaliculi).