The Fine Structure of the Eye of the Leech, Helobdella Stagnalis

J. Cell Set. a, 341-348 (1967) 341 Printed in Great Britain

THE FINE STRUCTURE OF THE EYE OF THE LEECH, HELOBDELLA STAGNALIS

A. W. CLARK* Department of Anatomy, University of Wisconsin, Madison, Wisconsin, U.S.A.

SUMMARY The eye of the rhynchobdellid leech, Helobdella stagnalis, has been examined with the electron microscope. The eye is composed of a cup of pigment cells surrounding a compact mass of photoreceptor cells. In addition to pigment granules, the pigment-cell cytoplasm is characterized by mitochondria, a Golgi complex, and profiles of rough-surfaced endoplasmic reticulum. The photoreceptor cell contains a microvillous rhabdomere. The microvilli arise from the membrane of a large intracellular vesicle and obliterate much of its lumen. No connexion between the lumen of the intracellular vesicle and the extracellular space has been observed. The plasmalemma of the photoreceptor cell is folded to form thin pleats of cytoplasm which separate adjacent receptor cells from each other. No glial-like cells have been seen in the receptor cell mass. Directly subjacent to the microvilli and surrounding the intracellular vesicle is a tortuous and predominantly smooth-surfaced endoplasmic reticulum. A pair of centrioles is found near the rhabdomere. The cytoplasm around the nucleus is characterized by smooth- and rough-surfaced elements of endoplasmic reticulum, many mitochondria, and a Golgi complex. Proximally, the receptor cell narrows to form a nerve fibre which joins those from other cells to form the optic nerve.

INTRODUCTION Leech photoreceptor cells aroused the interest of many classical cytologists and among a few of them (Whitman, 1899; Apathy, 1899) provoked a bitter and vitupera- tive controversy about the structure and derivation of this cell type. Hachlov (1910) was the last investigator of the classical period to deal with leech visual cells and, along with fresh observations of his own, he presented a comprehensive and critical survey of the literature. Of the three electron-microscopic investigations of leech eye structure, that of Hansen (1962) is the most comprehensive and encompasses the visual cell structure of seven species. Rohlich & Torok (1964) and Yanase, Fujimoto & Nishimura (1964) have restricted their investigations to the photoreceptor cells of a single arhynchobdellid leech, Hirudo medicinalis. The paper of Rohlich & Torok presents a detailed analysis of cell fine structure. In the following investigation the fine structure of the photoreceptor cell of the rhynchobdellid leech, Helobdella stagnalis, will be described and its significance discussed. • Present address: Department of Zoology, University of California, Berkeley, California, U.S.A.

Cell Sci. 2 342 A. W. Clark

MATERIALS AND METHODS Two species of rhynchobdellid leeches, Helobdella stagnate, and Placobdella rugosa, were used in this study. Leeches were fixed at room temperature in veronal- buffered i % osmium tetroxide at pH 7-7 (Palade, 1952) with the addition of o-ooi M CaCl2. After fixing for 1 h, tissues were rapidly dehydrated in a graded series of ethanols and then embedded in Epon 812 (Luft, 1961). Hardened blocks were sectioned on a Porter-Blum ultramicrotome using glass knives. Sections were picked up on parlodion-coated, carbon-reinforced grids, stained with lead citrate (Reynolds, 1963), and examined with an RCA EMU3E electron microscope.

RESULTS The eyes of Helobdella stagnate are found at the anterior end of the animal and occur as a single pair. Each eye consists of a cone or cup of pigment cells with the wide, open end of the cone pointing toward the surface of the animal. Within the pigment cup is a tightly packed conical mass of photoreceptor cells closely conforming in its shape to the inner surface of the pigment cup (Fig. 2) but separated from the pigment cells by a more or less uniform space 0-3-0-4 fi wide (Fig. 9). The pigment cup is composed of a single layer of cells, the nuclei of which are located along the outer surface (Fig. 2). The rest of each cell is composed of processes which are filled with pigment granules and run obliquely toward the cup's inner surface (Fig. 2). The cytoplasm of the pigment cell contains, in addition to the pigment granules, many profiles of rough endoplasmic reticulum, a prominent Golgi complex, and numerous mitochondria (Fig. 9). Each mature pigment granule is enclosed by a single membrane and, in H. stagnate, has a uniform outer diameter of about 0-35 fi (Figs. 2, 9). Close examination of pigment granules cut in grazing section reveals that they are composed of small particles, 40-50 A in diameter (Fig. 9, inset, arrow). Occasionally, pigment granules are found in the receptor cell mass, either in pigment cell processes or else free in the cytoplasm of the receptor cells. The photoreceptor cells of H. stagnate are large (the cell body being approximately 20 /i in diameter) and may be conveniently divided into several parts (Figs. 1, 2). The apical part contains the microvillous rhabdomere (Figs. 2, 4, 6) which.in many sections appears to be connected to the nuclear part of the cell by a narrow neck of cytoplasm (Fig. 1). Proximally, each receptor cell again narrows into a nerve fibre (Figs. 1, 8). This becomes grouped with fibres from other cells and bends over the lip of the pigment cup as the optic nerve (Figs. 2, 8). The plasmalemma of the photoreceptor cell has undergone extensive elaborations. One form of these can be seen in Figs. 1, 3, 4, and 6, where careful examination of adjacent cells reveals thin, intervening layers of cytoplasm. These pleats almost everywhere separate adjacent cells from each other and are seen to originate from the receptor cells themselves (Fig. 3). In spite of an extensive search, no glial-like cell has been detected in the photoreceptor cell mass. A second, much more extensive membrane elaboration is the microvillous rhabdo- Leech eye 343

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Submicrovillar retlculum

Fig. i. Simplified drawing of a photoreceptor cell from the eye oi Helobdella stagnalis, giving an approximate representation of its highly variable shape. Single arrows indicate the skein of filaments which runs the length of each microvillus. Double arrows indicate the neurofibrillar bundle. 344 A. W. Clark mere. The microvilli arise from the membrane of a large intracellular vesicle and project into its lumen. Although the microvilli obliterate much of it, the lumen of the vesicle is filled with a granular material (Fig. 4). A large number of sections has been examined for continuity between the intracellular vesicle and the extracellular space in the receptor cells of H. stagnalis and none was found. However, in view of the large size of these cells and the complex interrelationships of their membranes, such a continuity may have escaped detection. The microvilli are cylindrical in shape, approximately o-1 fi in diameter and o-6-i-4/i in length. Running down the centre of each microvillus for its entire length is a loose skein of filaments (Figs. 4, 10). The skein itself is approximately 100-130 A in diameter, and each filament is approximately 40-50 A thick. The rest of the lumen of the microvillus is filledwit h a slightly granular, electron-transparent material. Very often there is a slight constriction at the base of the microvillus (Figs. 1, 4, 6). Directly below the microvilli and surrounding the intracellular vesicle is an extensive and highly convoluted endoplasmic reticulum. Fig. 4 shows the submicrovillar reticulum in transverse section and Fig. 5 shows it in tangential section. In both figures, the cisternae have been artifactually distended and for the most part appear to be filled with an electron-transparent fluid. Scattered patches of granular material may also be seen. The reticulum membrane is predominantly smooth-surfaced, although a few ribosomes are seen to be associated with it in Fig. 5. The cytoplasm of the nuclear part of the cell is also characterized by a well- developed endoplasmic reticulum. Fig. 7 shows a region where there are distinct areas of both smooth- and rough-surfaced endoplasmic reticulum. There are several places (arrows) where the two types of endoplasmic reticulum can be seen to inter- connect. The cytoplasm of the receptor cell has a large number of mitochondria. They are found both in the submicrovillar cytoplasm and in the nuclear part of the cell, as well as in the nerve fibre (Fig. 8). A group of mitochondria is also seen in close proximity to a pair of centrioles. This grouping of mitochondria and centrioles is always located at the apical end of the cell, between the nucleus and the rhabdomere (Figs. 3, 6, 10). As in other reported examples, each centriole is composed of nine groups of three tubules arranged in a cylinder approximately 0-2 ji in diameter and 0-4/* in length. The centriolar microtubules are embedded in a finely granular matrix. Although it is not shown in any of the accompanying figures, an electron-opaque bundle of fibrils (Fig. 7) is often seen in the vicinity of the pair of centrioles. The bundle corresponds exactly in appearance to that seen in the nerve fibre (Figs. 1, 8). The receptor cell has a well-developed Golgi complex as well as many ribosomes unassociated with membranes. Numerous longitudinal and transverse profiles of microtubules are seen throughout the cytoplasm, especially in the nerve fibre (Fig. 8). In Fig. 5 microtubules are visible running parallel to the proximal surface of the submicrovillar reticulum (arrows). The cell nucleus is large and has a prominent nucleolus (Figs. 1, 2). The eye of the rhynchobdellid leech, Placobdella rugosa, has also been examined with the electron microscope. Although the pigment granules in the pigment cup are Leech eye 345 larger and more variable, and the receptor cells are larger, the fine structure of the eye is much like that just described for H. stagnalis. In Fig. 10 there appear to be three centrioles cut in oblique section, an arrangement encountered only once. Usually, only two centrioles are found and, as in Fig. 10, they are in close proximity to a group of mitochondria and the microvillous rhabdomere.

DISCUSSION The eye of Helobdella stagnalis has much in common with the eye of Hirudo medicinalis, the only other leech eye that has been studied intensively with the electron microscope (Hansen, 1962; Rohlich & Torok, 1964; Yanase et al. 1964). There are also some differences between the two eyes, however, and these may be a consequence of the relationship of the two animals. H. stagnalis and the Rhynchobdellidae may be less phylogenetically advanced than H. medicinalis and the Arhynchobdellidae (Mann, 1962). The eye of Hirudo is larger than that of Helobdella. The difference in size is accounted for both by the greater size of the Hirudo photoreceptor cell and by the looser packing of the cells. Hirudo receptor cells are separated from each other by a uniform space, easily seen with the electron microscope. From the description of Rohlich & Torok it is clear that each cell is endowed with a sheathing of cytoplasm derived from what presumably is a glial cell. Although the sheathing is not complete, much of the surface of the receptor cell is covered. In contrast, no glial-like cells have been detected in the Helobdella eye after the examination of many sections from several eyes. Instead, the receptor cells are tightly packed together and have pleats of then- own cytoplasm separating adjacent cells from each other. These pleats may serve the photoreceptor cells as a form of self-sheathing in lieu of the glial-like cells found in the presumably more highly evolved Hirudo eyes. The greater size of the Hirudo receptor cell is partly accounted for by the larger intracellular vesicle into which the microvilli project. Unlike Helobdella, where the microvilli obliterate much of the vesicular lumen, the microvilli in the Hirudo eye form what resembles a brush border around the inner margin of the vesicle. In Hirudo, the vesicle is large and is filled with a flocculent, electron-transparent material. The smaller size of the intracellular vesicle in Helobdella may be related to the tight packing requirements of the photoreceptor cell mass. Rohlich & Torok interpret as vesicles the large number of circular membranous profiles they found in the cytoplasm of the Hirudo receptor cell. I suggest, however, that the profiles represent transverse sections through a tortuous, smooth endoplasmic reticulum and therefore correspond to the smooth reticulum found in the Helobdella receptor cell. None of the investigators of the Hirudo receptor cell has reported the presence of centrioles. However, it seems likely that the large size of these cells as well as the looser packing of the cell mass reduce the chances of encountering this small organelle in the electron microscope. It is predicted that a pair of centrioles in close association with the microvillous rhabdomere will be found in Hirudo and in arhynchobdellid leeches generally. 346 A. W. Clark The receptor cells of Hirudo and Helobdella resemble each other closely in having a large number of mitochondria and a well-developed endoplasmic reticulum. In parti- cular, they both have a distinctive reticulum which surrounds the intracellular vesicle and is located immediately subjacent to the microvilli. The receptor cells in both are characterized by electron-opaque bundles of fibrils frequently encountered in the cytoplasm around the nucleus. It is believed that these bundles are continuous with those found in the nerve fibre and are neurofibrillar bundles, like those observed in other parts of the leech nervous system by Gray & Guillery (1963). Finally, there appears to be no connexion between the lumen of the intracellular vesicle and the extracellular space in the photoreceptor cells of either genus. Current generalizations on photoreceptor cell fine structure predict the presence of a large number of mitochondria and a great elaboration of cell membrane. The Helobdella receptor cell is congruent with such notions. Eakin (1965) has generalized further about the ciliary or non-ciliary derivation of the membranous elaboration. According to his theory, the type of derivation may be a reflexion of the phylogenetic origins of the animal bearing the cell. However, recent papers by Dhainaut-Courtois (1965), Fischer & Brokelmann (1966), Kern&s (1966), and Krasne & Lawrence (1966) indicate that the derivation of the membranous organelle in annelid photoreceptor cells is more complex than was first imagined. In the eyespot of Branchiomma vesiculosum examined by Krasne & Lawrence, there is a stack of membranous discs, each of which is derived from a ciliary membrane. On the other hand, the receptor cell of Nereis pelagica (Dhainaut-Courtois, 1965) has a large number of microvilli and these also appear to be derived from a ciliary membrane. Nothing resembling a cilium has been observed in the Helobdella eye. Neverthe- less, in view of the occurrence of cilia in polychaete eyes, the presence of a pair of centrioles close to the microvilli in the receptor cells of both H. stagTiaUs and P. rugosa deserves comment. Sorokin (1962) has shown that rudimentary cilia are regularly encountered in differentiating fibroblasts and smooth-muscle cells from neonatal chicken and mammalian tissues. Since they are so rarely encountered in the adult tissues, the cilia may be shed as differentiation proceeds. Pollister (1933) has shown that in amphi- bians many non-ciliated epithelial cell types have centrioles associated with their apical surfaces. Consequently, the pair of centrioles in the Helobdella receptor cells may be all that remains of a rudimentary cilium which was shed early in the cell's development. This would be Eakin's interpretation, since a very similar sequence appears to occur in the eye of the snail, Helix aspersa (Eakin & Brandenburger, 1967). Even more simply, a cilium may never be involved with the rhabdomere in Helobdella. An alternative explanation might account for the coincidence in the location of this pair of centrioles and the role of these organelles in other photoreceptor cells. According to LeBoucq (1909), Seefelder (1910), De Robertis (1956), Tokuyasu & Yamada (1958), Carasso (1959), Dowling & Wald (i960), Ueno (1961), Wald, Brown & Gibbons (1962) and Cohen (1963) the pair of centrioles is the Crucial initiating element in the differentiation of the vertebrate rod and cone outer segments. Perhaps the centrioles serve a similar function in the photoreceptor cells of Helobdella and Leech eye 347 Placobdella and induce the elaboration of the microvillous rhabdomere. According to this view, the cilium and its modifications found in the polychaete eyes mentioned above may be only one of the membrane elaborations presided over by centriolea. A resolution to these questions may lie in a study of the developing eye of the leech. This is at present under investigation.

The author is greatly indebted to Dr D. B. Slautterback for his advice and patience during the course of this investigation. The author also wishes to thank Dr R. M. Eakin for his very useful criticism of the manuscript. This investigation was supported by Public Health Service Grants nos. GM 723-04 and NB 34,870-01 of the National Institutes of Health.

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(Received 13 January 1967)

Fig. 2. A low-magnification electron micrograph of the eye of H. stagnalis which shows the cup of pigment cells and the tightly packed mass of photoreceptor cells. In this section, many receptor cell nuclei (n) and one microvillous rhabdomere (r) may be seen, (mu, muscle cell; on, optic nerve.) x 3200. Journal of Cell Science, Vol. 2, No. 3

A. W. CLARK (Facing p- 348) Fig. 3. An electron micrograph of the eye of H. stagnalis, showing portions of several receptor cells and a microvillous rhabdomere in transverse section. Arrows indicate pleats of receptor cell cytoplasm which separate adjacent cells from each other. The origin of one of these pleats from the receptor cell may be seen at the top of the figure between the two arrows. The double arrow indicates a centriole in transverse section. (gc, Golgi complex; m, mitochondria.) x 21000. Journal of Cell Science, Vol. 2, No. 3

A. W. CLARK Fig. 4. An electron micrograph which shows a longitudinal section through a rhabdomere in the eye of H. stagnalis. Single arrows indicate the skein of filaments that run the length of each microvillus. The double arrow indicates a pleat of receptor cell cytoplasm, (en, cisternae of the submicrovillar reticulum.) x 35 coo. Fig. 5. An electron micrograph of the eye of H. stagnalis. In this section the submicrovillar reticulum has been cut tangentially. Careful examination of the reticulum membrane reveals scattered ribosomes attached to it. Arrows indicate microtubules running parallel to the proximal surface of the reticulum. (en, cisternae of the submicrovillar reticulum.) x 28000. Fig. 6. Electron micrograph of the eye of H. stagnalis, showing parts of several receptor cells. In close proximity to the centriolar pair is a group of mitochondria (m) and the microvillous rhabdomere (mv). Arrows indicate pleats of receptor cell cytoplasm separating adjacent cells from each other, x 34000. Journal of Cell Science, Vol. 2, No. 3

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A. W. CLARK Fig. 7. Electron micrograph of the eye of H. stagnate which shows the cytoplasm of the nuclear part of the receptor cell. Arrows indicate points of junction between the smooth- (ser) and rough-surfaced (rer) endoplasmic reticulum. Note the polysomes in the reticulum. («/, neurofibrillar bundle.) x 29000. Fig. 8. Electron micrograph of the eye of H. stagnalis which shows a receptor cell narrow- ing at its proximal end to form the nerve fibre (asterisks). Oblique sections of several other nerve fibres are visible. Arrows indicate microtubules and double arrows indicate sections of neurofibrillar bundles, (n, nucleus of the receptor cell.) x 15000. Fig. 9. Electron micrograph of the eye of H. stagnalis, showing part of a pigment cell and a small portion of an adjacent photoreceptor cell (re). The inset reveals the parti- culate nature of the pigment granules (arrow), (gc, Golgi complex ;n, nucleus.) x 27000; inset x 49000. Fig. 10. Electron micrograph of the eye of P. rugosa showing a part of a single receptor cell. Three centrioles appear to have been sectioned obliquely. As in Fig. 6, the centrioles are in close proximity to mitochondria (m)and the microvillous rhabdomere. Arrows indicate transverse sections of the skein of filaments which run the length of each microvillus. x 36000. Journal of Cell Science, Vol. 2, No. 3

A. W. CLARK