Cell and Tissue Research

Cell Tissue Res. 198, 501-520 (1979)

9 by Springer-Verlag 1979

Membrane Specializations in the Peripheral Retina of the Housefly Musca domestica L. Che Chi, Stanley D. Carlson, and Richard L. St. Marie* Department of Entomology, University of Wisconsin Madison, Wisconsin 53706, USA

Summary. Membrane specializations of the peripheral retina of the housefly (Musca domestica) are revealed in thin sections and freeze fracture/etch replicas. Septate junctions are abundant in corner areas of the pseudocone enclosure bonding: between homologous corneal pigment cells (CPC); between homologous large pigment cells (LPC); between CPC-LPC; between Semper cells (SC); between SC-CPC. Spot desmosomes are present between Semper cells. It is likely that septate junctions function as strengthening adhesions in this area. A new m e m b r a n e specialization similar to a continuous junction was observed between retinular cells of the same or adjacent ommatidium. This junction has indistinct septa in the 115A intermembrane cleft and is intermittent in character. When this junction is absent, the apposed cells gape apart. In freeze fracture studies, this junction is characterized by bridges composed of fused membrane particles and randomly arranged particles on the P face, and noncorresponding grooves on the E face. The ridges are elongate and roughly parallel and sometimes they form enclosures. Mitochondria line up along these junctions, often within 90A of the unit membrane. This membrane specialization has characteristics of tight and continuous junctions. In line with previous findings, we suggest that this junction assists in retinular cell orientation, possibly in enforcing the ommatidial twist and in maintaining localized ionic concentration gradients between retinular cells. Key words: Peripheral retina - Transmission electron microscopy - House fly M e m b r a n e specializations and pigment cells - Photoreceptor cells. Send offprint requests to: Dr. StanleyD. Carlson, Department of Entomology, Universityof Wisconsin, Madison, Wisconsin 53706, U.S.A. * We gratefully acknowledgesupport from the N.I.H., National Eye Institute, EYO-1686and from the Collegeof Agricultural and Life Sciences, Hatch Project 2100. Dr. Tom Reese and Dr. John Heuser assisted one of us (RLSM) in freeze etch technique at the Neurobiology Training Course, Marine Biology Laboratory, Woods Hole, Mass. Support for RLSM at MBL came from a Grass Foundation Fellowship. This work was also supported in part by Grant RR 00167 from the N.I.H. to the Wisconsin Primate Center. We heartily thank Dr. Philippa Claude, Primate Center, UW, Madison, for training in the freeze-fracturetechnique and for criticallyreading this manuscript. Dr. Robert Goy, Director of the Primate Center, is acknowledged for his kind permission to use the Center's freeze etch apparatus. Professor Hans Ris, Department of Zoology gave permission for use of the high voltage electron microscope. Mr. Martin B. Garment provided able darkroom assistance.

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Histological interest in the dipteran eye dates back at least 100 years (Grenacher, 1879; Dietrich, 1909) and the major emphasis has been on the better visualization of the ommatidium and its rhabdomeres (Goldsmith and Philpott, 1957; TrujilloCenoz, 1965; Boschek, 1971). Up to the present there has been a general lack of knowledge about the membranes of the photoreceptor cell other than those of the rhabdomeric microvilli which contain the visual pigment. Non-rhabdomeric membrane of the fly photoreceptor cell apparently contains visual pigment as well (Schinz et al., 1978). Seven different cell types are present in the peripheral retina and there are at least 10 different cell-to-cell juxtapositions. Little attention has been paid to intercellular contacts in this epithelium. Only a few membrane specializations have been described in the retinal epithelium, viz., belt desmosomes between contiguous retinular cells (Trujillo-Cen6z, 1965; Boschek, 1971), septate junctions between the various pigment cells (Chi and Carlson, 1976), gap junctions between retinular axons (Simpson and Shaw unpublished, cited by Shaw, 1977), chemical synaptic specializations at the retinular axons terminals (Trujillo-Cen6z, 1965), and close appositions with interdigitating photoreceptor axons (Chi and Carlson, 1976b). A number of vital functions are made possible in an epithelium equipped with appropriate intercellular junctions, viz., cell-to-cell cohesion and spacing, solute chanelling between cells, diffusion barriers and electrical synapses. The structure and function of membrane specializations in insects and other animals have been reviewed by Harvey and Blankemeyer (1975), Staehelin (1974), Staehelin and Hull (1978), and Satir and Gilula (1973). This study has two major goals: (1) to identify junctions in the peripheral retina based on several criteria, and (2) to evaluate their functional/structural significance in the compound eye. In the first instance, data from the freeze fracture technique complement our understanding of the eye as based on thin sections. Both methods help to assess the nature of a particular cell-to-cell contact. Freeze fracture replicas offer vistas over many square microns of cellular surface. On the other hand, thin sections are samples involving fractions of microns. In the latter technique, tannic acid was used as a mordant between OsO 4 and lead salts which resulted in higher contrast in both intra- and extracellular structures. Simonescu and Simonescu (1976a, b) reported that tannic acid causes the tissue to be more resistant to extraction and deterioration of structural detail. These last virtues may be the reason for our visualization of the junction between photoreceptor cells. Such data add to a more complete concept of the functional organization of the compound eye.

Methods and Materials

For transmission electronmicroscopy(thin sections)compound eyes werehalved and fixed for 1.5 to 3.5 h in 2.5 ~ glutaraldehydein 0.1 M phosphate buffer.The tissue was then washedin three changesof buffer (30 min each) and postfixed in 2~ osmium tetroxide in 0.1 M phosphate buffer for 1.5h. Conventional ethanolicdehydration and embedding(Spurr| proceduresfollowedas detailed by Chi and Carlson (1976). Tannic acid, AR, from Mallinkrodt, Inc., St. Louis, Mo. was either added to glutaraldehydeor applied after osmium tetroxide. In the former case, 4 ~ tannic acid was added to a solution of 2.5 ~o

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glutaraldehyde in 0.2 M cacodylate buffer (pH 7.1). The tissue was then washed in 0.1 M cacodylate buffer, postfixed in 2 % osmiumtetroxide in 0.1 M cacodylatebuffer, and dehydratedin a gradedethanol series. When tannic acid was not mixed with fixative the fixed tissue was exposed to a 1% solution of tannic acid in 0.05 M phosphate buffer for 40 rain after OsO4f'Lxation,then washedin 1% sodiumsulfate in 0.05 M phosphate buffer for 5 min. Subsequent procedures were the same as described above. For more information about technique and theory of tannic acid usage in TEM see Simonescu and Simonescu (1976a, b) and Van Deurs (1975). In freeze fracture studies, whole or hemisected eyes were fixed for 2-3h at 4~ in 2.5% glutaraldehyde in phosphate, cacodylate or Hepes buffer (pH 7.2). Tissue was then infiltrated with glycerol in an ascending concentration series to 25 % glycerolin buffer and frozen on gold or copper specimenholders in liquid freon cooledin liquid nitrogen. Specimenswere fractured in a Balzers 301 or 510 freeze-etch machine at -115~ and then coated with platinium and carbon. Replicas were subsequently cleaned in hypochloritebleach and picked up on formvar-carboncoated slot grids and viewed in a Phillips EM 210 or AE1 6B. One micrograph of thick sectioned material (0.3 Inn) was taken in the AE1 7 High Voltage Electron Microscope, University of Wisconsin, at 1000kV.

Results The retinal epithelium of the housefly is an association of seven different cell types some of which are connected to four noncellular entities: interommatidial space, lens, pseudocone and basement membrane (basal lamina). From this view the reader can begin to visualize the peripheral retina of the housefly as a fanned out tuft of columnar photoreceptor (R) cells organized into 8celled clusters, each called a retinula. Three types of screening pigment cells are draped above and around various portions of the retinula. Six to eight large pigment cells (LPC) contact the proximal margins of a single lens facet then branch into attenuated processes that are linked with each other laterally. Distally, large pigment cells enclose the pseudocone for a few microns and extend like parachute cords around the chalice-shaped pseudocone and retinula. This pigment sheath extends to the basement membrane. Consequently the large pigment cells are the principal suspensory and optical shielding element for the encased retinula. Several microns below the lens two corneal pigment cells (CPC) take over as the main lateral boundary cells for the pseudocone and Semper cells (SC). Proximally, this pair of CPC clasps the distal retinula. The pseudocone is supported at its base by a cushion of four conjoined SC. Small pigment cells are located near the basement membrane and indent slightly into the interommatidial cavity (space). The last elements to be considered part of the retinal epithelium are the large bore tracheae that penetrate the basement membrane from below and a single branch of which usually ascends along each retinula. In the survey (freeze fracture) micrograph (Fig. 1) with its low magnification it is difficult to discern the various membrane specializations. Nevertheless, low angulated ridges (rows of membrane particles) can be resolved on the P (or cytoplasmic fracture) face of an R cell contacting a pigment cell (area outlined in the frame). This field is seen at higher magnification in Fig. 11. The Semper cell surface (P face) shows swirling rows of membrane particles underlying the extensive septate desmosomes (see also Figs. 7-9). In the distal retinal epithelium, LPC's and CPC's surround each pseudocone

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Fig. 1. Freeze-fracture replica of retinal epithelium. Under the corneal lens (L) is pseudoeone (PC) which rests on four Semper cells (SC). Rhabdomeric microvilfi (r) of peripheral retinular cells (R) are directly below Semper ceils. Superior central cell (*) is in center of figure. Septate junctions between Semper cells are in middle ofmicrograph (Fig. g is enlargement of this area). Large nucleated (N) pigment cells (/.,PC) laterally shroud each ommatidium. Boxed area shows membrane specializations between retinular cell and large pigment cell (see also Fig. 11). Encircled arrow gives direction of platinum deposit in all figures. • 4200

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isolating them from each other. The LPC's are sandwiched by the CPC's throughout most of the length of the pseudocone (Fig. 2). This cellular laminate is held together by septate junctions between the LPC's and CPC's making up the pseudocone enclosure as well as welding together contiguous pseudocone enclosures. Specifically, septate junctions are found between LPC's, between CPC's, and between CPC and LPC (Figs. 2, 3). Septate junctions are also found between SC's and intermediate to SC's and CPC's (Fig. 6). Transverse thin sections (Fig. 2) show electron dense septa which span an intercellular cleft of about 125A. These intercellular bridges measure ca 95A in thickness with a center to center spacing of 210-250A. In tangential sections a honeycomb-like pattern emerges and the electron lucent circular areas are about 120 A in diameter. Gap junctions (Figs. 4-5) are noted between pigment cells in the pseudocone area. Freeze fracture replicas (Fig. 5) show plaques of membrane particles on the E face that are indicative of gap junctions. Various numbers of membrane particles, randomly packed, are usually found in a plaque on the E face. Particle diameter is 100-135 A. In a rare case, a patch of particles is found on the P face of a pigment cell near the distal retinula (Fig. 3). Our recent lanthanum tracer studies (unpublished) have revealed extensive gap junctions between pigment cells. This finding supports the present freeze fracture and HVEM (Fig. 4) results. Cross sections of Semper cells (Fig. 6) show septate junctions between them. Also, in this particular region, spot desmosomes intervene in areas where septate junctions are absent. The unspecialized cell apposition represents a minority condition. Adding to the cohesiveness of this 4-cell unit is the interdigitation of the Semper cells with each other (Figs. 6, 8) particularly near their exterior margins where no membrane specializations are present. Where the Semper cell is laterally encased by a corneal pigment cell, septate junctions are observed (Fig. 6) in thinsectioned tissue. Septate junctions are also revealed in freeze fracture planes between Semper cells (Fig. 7) and between Semper cells and corneal pigment cells (Figs. 8, 9). Particles seen on the Semper cell's plasma membrane (P face) are usually found in numerous parallel tracks which form gently undulating girdles or more accentuated loops ("thumb print whorls") which encircle members of this quartet of cells. Patches of particles seen on Semper cell P face membrane (Fig. 7) may represent the spot desmosomes seen in thin section (Fig. 6). It is generally agreed that the intramembranous particles are aligned with the intermembranous septae. If so, the dimensions and packing pattern of the undulating ranks of membrane particles seen in freeze fracture (FF) preparations should generally correspond to the honey comb pattern of the intercellular septa as revealed in thin sections (TS) when this desmosome is sectioned tangentially. Based on the previously mentioned TS data, center to center spacings between clear circular areas (honey-comb) average 230_+ 20A a value which relates well to that measurement (200 _+60A)between adjacent membrane particles in a row. Particle diameter (FF) is about 122 A which correlates with a diameter of 120 A for the clear circular areas (TS tangential). Some variability exists in distances between particles of a given line on the replica surface, particularly near the end of the rank and in fairly straight rows.

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An unusual junction, reported for the first time, is found between retinular cells of an ommatidium as well as between contiguous retinular cells from adjacent ommatidia (usually R 2 of one ommatitium and R 5 of the adjoining ommatidium) (Figs. 10, 13-23). Both P (cytoplasmic) and E (external) faces of retinular cells have been exposed to reveal a series of ridges (rows of aligned membrane particles) on the P face and grooves on the E face. Loose, unoriented particles abound between ridges and both discrete and fused (into ridges) particles are about 90-100A in diameter. Ridges on the P face only occassionally correspond to grooves on the E face of the apposed cell (Figs. 10, 13). Most ridges are roughly parallel to the R cell's long axis with little tendency to form the polygonal enclosures characteristic of tight junctions, (Figs. 10, 13, 15). But in some cases circular ridges (Fig. 16) near the area of the basement membrane are nearly continuous and enclose appreciable areas of the R cell's intramembranous surface. A more loosely-knit organization of particle ridges (Fig. 14) is also present on R cells. In this case particle rows are short, without orientation or connections to each other. This pattern is mimicked by the tracheal cell membrane where it contacts a retinular cell (Fig. 12). Another variant of this system between R cell and large pigment cell (Fig. 11) shows highly angulated ridges forming rectangular and trapezoidal figures. The thin section correlate of this membrane specialization has been diligently sought. In the region where apposed R cells exhibit this specialization in freeze fracture replicas, thin sections reveal indistinct septa associated with fine granular material in the intercellular space (Fig. 23). The distinctness of the septa in this junction might depend on the plane of section or viewing angle. This has sometimes been demonstrated by viewing, then tilting, thick sections on the stage of a high voltage electron microscope (Chi, unpublished). Nevertheless, some mystery remains in that septate junctions in the distal retinula are usually crisp in outline while the retinular cell junctions are never resolved with the same clarity. The most intriguing feature of this junction are the nearby mitochondria. These organelles nearly touch the plasma membrane (within 90A of the retinular cell's membrane) and are nearly always aligned parallel to the cell membrane (Figs. 18, 21-23). Often sinuous mitochondria follow the plasma membrane's contours and in

Fig. 2. Interstitial area of pseudocone (PC) enclosure made up of large (LPC) and corneal (CPC) pigment cells. Note septate junctions between CPC and LPC, between LPC's and between CPC's (arrow) and tangentially sectioned septate junctions (honeycomb?) in far upper right corner (double arrow). x 22,850 Fig. 3. Freeze fracture replica. Distal retina. Membrane particles aligned in parallel ridges on P face (P) of distal pigment cell. Particles in rows comprise septate desmosome which connects 2 pigment cells. Note patch of particles (arrow) on pigment cell membrane P face. x 24,750 Fig. 4. Thick section: HVEM. Small processes of two large pigment cells make contact near level of LPC nuclei. This apposition is probably a gap junction based on our (unpublished) tracer studies, x 14,000 Fig. 5. Three patches of membrane particles on E face of large pigment cell probably are sites of gap junctions. These specializations are slightly proximal to cell body of LPC and in same general area as that of Fig. 4. • 26,500

Fig. 6. Cross section through portions of four Semper cells (SC) and part of an enclosing corneal pigment cell (CPC). Semper ceils have undulating surfaces (upper left corner) which infold (*) into each other. Sept.ate junctions are on all interior border membranes of Semper cells (single arrows) and between Semper cell and corneal pigment cell (CPC) (double arrows). Membrane thickenings between apposed Semper cell membranes are probably spot desmosomes (D). Numerous cross-sectioned microtubules in Semper cells. N nucleus. • 21,000

Fig. 7. Swirling rows of discrete membrane particles on P face membrane of Semper cell show extent of septate junction between two Semper cells. Microvilli seen on left border pseudocone. Circular aggregations (D) of membrane particles correlate with spot desmosomes seen in thin section (Fig. 6). This field is part of Fig. 1. x 35,000 Fig. 8. Cleavage plane through base of pseudocone (PC) revealing P face of external surfaces of three Semper cells (SC) and E face of neighboring corneal pigment cell (CPC). Wavy, parallel rows of membrane particles on P face of Semper cells show extent of septate junctions between Semper cell and overlying corneal pigment cell. Semper cells interdigitate with each other (*). x 19,500 Fig. 9. Detail of Fig. 8. Parallel rows of membrane particles on P face of Semper cells indicative of septate junctions. • 400,000

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some cases an R cell membrane will form a finger-like evagination, even doubling back on itself (Fig. 22). In this case a mitochondrion has partly entered a cytoplasmic herniation and thus maintained close contact with two sides of the R cell process. This extrusion of R cell membrane and mitochondrion gives the cell greater surface area over which to contact its neighbor. The functional consequences of such a concentration of mitochondria in general, and specifically of two mitochondria in the same cell sandwiching the plasma membrane, will be discussed later. In the immediate area of close apposition of mitochondrion and plasma membrane one finds elements of the rough endoplasmic reticulum and aggregations of free ribosomes (Figs. 18, 21). Spherical or eliptical vesicles of up to 2500A in diameter occasionally accompany the grouping of mitochondrion - plasma membrane - endoplasmic reticulum. Another salient feature of this junction is its peculiar discontinuous nature. In thin section, at intervals of 0.5 to 2 txm, the apposing unit membranes show a cleft as wide as 100-200 nm for a distance of 300 nm or so before closing to the usual 100115A interspace (Figs. 18, 21, 22). An intriguing feature of this junction is that there may be some specialized association between mitochondrion and plasma membrane. Occasionally, in thin section, electron dense material is observed spanning the space between the plasma membrane and the mitochondrion (Figs. 18, 22). Freeze fracture data suggest the possibility of a physical association between mitochondrion and unit membrane of the R cell. We interpret the kidney shaped membrane remnant (Fig. 20) as a fragment of the outer leaflet of a mitochondrion. We speculate that the bond between mitochondrion and plasma membrane was tenacious enough to coerce the cleavage plane directly from the plasma membrane of the neighboring cell, to the membrane of the mitochondrion.

Fig. 10. Fracture face of superior central retinular cell (R7) that nestles between two peripheral retinular (R 1 and R6) cells. Numerous membrane particles on P face of g 7 and R 6. Some particles fuse into longitudinal ridges. E face of R 1 shows longitudinal grooves. Grooves seldom correspond to ridges. Gutter (G) on lateral surface of R 7 (between R 7 and R1) accomodates Semper cell process (Sp). On either side of gutter note single line of particles. Area (P face) through which belt desmosome (*) extends has scant particles. Rhabdomere (r) of retinular cell (R1) at lower right, x 37,500 Fig. 11. Fracture plane between retinular (R) and large pigment cell (LPC). Numerous membrane particles on P face of R cell align to form angular ridges occassionally connecting with other ridges to form partial enclosures. No order exists among membrane particles in interridge area and no corresponding pits are found on the E face of LPC. • 40,000 Fig. 12. Membrane overlying trachea (7); membrane particles aligned in short rows on P face without continuity or special orientation. Upper right of figure is E face (E) ofretinular cell membrane, x 40,000

Fig. 13. Membrane specialization between two retinular cells. Intramembrane panicles on P face (P) arrayed in ridges roughly parallel to long axis of R cell. Grooves are on E face. Semper cell process (S) projects between two apposed retinular cells. Small portion of E face membrane of retinular cell adheres to distal portion of Semper cell process. Vertical row of particles projects beside Semper cell process. A particle free zone (*) on P face of retinular cell underlies belt desmosome, x 40,000

Fig. 14. Retinular cell. Membrane particles on P face organized into abbreviated rows (short ridges) without apparent orientation. Long axis of R cell in Figs. 14-16 is 3-9 o'clock, x 38,000 Fig. 15. P face of retinular cell. Membrane particles mainly arrayed in longitudinal ridges (L) parallel to long axis of R cell. Circumferential ridges (C) meet longitudinally disposed rows and, in some cases, form nearly complete rectangular enclosures. Semper cell process cleaved away leaving longitudinal depression in retinular cell and 2 remnants (S) of Semper cell membrane. Area (*) of belt desmosome shows no particles, x 38,000 Fig. 16. Replica. Retinular cell near basement membrane with adjacent trachea (7). Aligned membrane particles on P face of R cell form circular, nearly enclosed, ridges which grade into series of longitudinal ridges (arrows) x 38,000 Fig. 17. Higher magnification from Fig. 16 showing randomness of interridge membrane particles and polygonal enclosures formed by intersecting ridges. All particles of similar diameter, x 94,500

Fig. 18. Apposed plasma membranes of two retinular ceils. Indistinct septa span intercellular cleft. Mitochondria parallel to, and in close contact with, plasma membrane. Endoplasmic reficulum (ER) and free ribosomes (R) in vicinity of mitochondria. Figs. 18, 21-23 are tannic acid preparations, x 40,500 Fig. 19. Fracture face of retinular cell; counterpart to Fig. 18. Numerous particles randomly distributed on P face (P); some aligned into elongate ridges. E face membrane shows grooves. Abundant mitochondria (M) aggregate along cell border, x 42,000 Fig. 20. Cleavage between 2 retinular ceils. Possible fragment of outer leaflet of mitochondrion (M) adheres to a retinular cell which, for the most part, has been cleaved away. Adhesion between mitochondrion and retinular cell membrane may be appreciable so that cleavage plane went from membrane of one cell through membrane of second cell to membrane of mitochondrion; E face (E); P face (P). x 38,000

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Discussion Specialized areas of contact between adjacent cells are extremely important features in any assemblage of cells. It is not surprising that a variety of membrane specializations exist in the peripheral retina of the fly's eye. However, the occurrence of specialized junctions between photoreceptor cells was unexpected. Septate Junctions. These were first described among invertebrates in Hydra by Wood (1959). In 3-dimensional perspective each intercellular "bridge" or septum is but the end-on profile of an elongate corrugated ("pleated") sheet (Satir and Gilula, 1973), or the septa may be manifested in a hexagonally structured array of folds termed "honey comb" (Danilova et al., 1969), which was first observed in silkworm gut epithelium. As both honey-comb and pleated-sheet types of septate desmosomes have been found by us between Semper cells and pigment cells we cannot stipulate the type. Objection to such a distinction by Staehelin (1974) and Satir and Gilula (1973) is based on the fact that both types occur together. Septate desmosomes very likely impart structural cohesiveness between pigment and Semper cells near the distal retinula. Such a function is consonant with our previous finding (Chi and Carlson, 1976) that the whole ommatidium is suspended from the lens by large pigment cells while a second "sling" of corneal pigment cells grasps the large pigment cells, most of the dioptric apparatus and distal retinula. Septate junctions are numerous between heterologous and homologous pigment cells particularly in the interstitial region between three or four adjoining ommatidia. The rows of discrete particles on the exposed P face of the membrane may be the underpinnings of septa. According to a model by Satir and Gilula (1973), particle and septum forge a link that bonds the two leaflets of one plasma membrane to those of the contiguous cell is plasma membrane via the intermembranous septa. If that is so one would expect to see the undulation period equal the center-to-center spacing between membrane particles in adjacent rows. What we find instead is that the distance between membrane particles in neighboring rows is quite variable and often exceeds the lattice constant of the intercellular septa seen in thin sections. This disparity is in line with the findings of Noirot-Timothre et al. (1978) on septate junctions in the epithelium of insect hindgut. Nevertheless, the remarkable correlation between septa/particle lattice constants and particle vs clear circular space diameters in a single row is noteworthy

Fig. 21. Thin section of junction between two retinular cells of same ommatidium. Space between apposed plasma membranes alternately expands (arrows) and constricts. Narrowed intermembranal space has irregular, indistinct septa, x 38,000 Fig. 22. Plasma membraneof retinular celldoublingback on itself. Membranemakescontact with itself (arrow) and with neighboring retinular cell (double arrow). Mitochondrion (M) follows membrane contour and herniates into evagination. Note proximity of apposed plasma membrane to all mitochondria, x 51,000 Fig. 23. High magnificationof apposed plasma membranesbetweentwo retinular cells. Indistinct septa and fine granular material appear in interspace (about 115A wide). Mitochondrion (M) nearby. ER endoplasmic reticuhim, x 185,000

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and shows the commonality of some features of septate junctions as demonstrated by two (TS and FF) very different techniques. With the emerging concept of a retina-lamina barrier (similar to the "bloodbrain" barrier) in the locust compound eye (Shaw, 1975, 1977, 1978) with its meager extracellular space, septate junctions "... may reasonably be considered as limiting a complex system of channels which considerably retard the diffusion of fluids and solutes in the intercellular space. These junctions would thus play a role of permeability barrier, fulfilled in epithelia of vertebrates by the tight junctions ..." (Noirot-Timothbe et al., 1978). A paper in preparation (St. Marie, Chi and Carlson) will cover in more detail the anatomical correlates of this barrier. Suffice it to say, that these collective septate junctions located in the very distal portion of the retinal epithelium might be considered an apical barrier retarding or occluding movement of ions and small molecules. Gap Junctions. Freeze-fracture data, e.g., particles aggregates, particle location on E face, particle disorganization within the plaque, and lattice constant, leave little doubt that gap junctions are present between pigment cells in the fly retina. The gap junction or nexus is considered to be a channel for intercellular communication; gap junctions are found between nonexcitable cells that are electrically coupled, between electrically coupled neurons and between cells that are metabolically united (cf. Staehelin, 1974). There is nothing known about pigment cells in insect retinas that requires intercellular communication. One can speculate that biosynthetic products of screening pigment granules might advantageously circulate among the distal pigment cells. The copious lipid inclusions with associated mitochondria and gap junctions of the large pigment cells may, in concert, be involved in providing retinal or other visual pigment precursors to the photoreceptor cells. Recent evidence (Yoshikami and Nrll, 1978) shows that liposomes of skate retinas are suppliers of retinal to the photoreceptor cells. Specifically, the liposomes "protect" and "deliver" vitamin A in the retina. How could such a system work in the fly if the gap junctions, supposedly mediating this transport, were only between pigment cells and not between pigment cells and photoreceptor cells? Our freeze fracture evidence is ambiguous on this point. In many cases we note gap junctions on pigment cells but cannot determine if these communicate with another pigment cell or a contiguous photoreceptor cell. No thin section findings are as yet available on this point from our laboratory or from others. A final clue to the understanding of the function of gap junctions between pigment cells comes from knowledge (cf. Schwartzkoff, 1974; Thurm, 1970) that certain accessory cells intimately associated with insect chemo- and mechanoreceptor neurons are responsible for maintaining ionic gradients that make up the transepithelial potential. Pigment cells electrically coupled to themselves might fulfill this function. Retinular (R) Cell Junctions. Junctions between photoreceptor cells or between receptor and first order interneurons should be functionally important. As examples, septate junctions are present between photoreceptor ceils of the leech (Lasansky and Fuortes, 1969) and gap junctions exist between receptor (rod) terminal telodendria in the toad (Fain et al., 1975) and fish (Witkovsky et al., 1974). Gap junctions are likely sites for electrical conduction but no evidence currently

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exists for their presence between retinular cells in the peripheral retina of flies. The curious "zipper-like" junctions between retinular cells of the mutant (x-12) Drosophila (Alawi et al., 1972) are rarely seen in Musca. (The function of this junction is not known directly, but Alawi and co-workers suppose it is related to the altered resting potential and lack of electrical response of R 1-6 in this particular mutant of Drosophila). The newly described intermembranal specialization between fly retinular cells, as yet unnamed, does not fit any precisely recognized type of membrane specialization, although it is similar to both tight and continuous junctions. R cell junctions bear some likenesses in thin section to the continuous junctions first described by Noirot and Noirot-Timoth~e, (1967); for example as to the width of the intercellular space (ca l10-115A) and presence of a fine granular material (probably glycoprotein, Dallai, 1975) that is organized into faint or indistinct septa of variable periodicity. Dallai (1975) states that intermembranous septa are not visible "... after the usual staining" (our italics). Certain freeze fracture characteristics of R cell junctions are also consistent with those of continuous junctions, e.g., particles aligned in straight or variously curved ridges on the P face, with the particles in the interridge area being extremely numerous but unorganized. Known continuous junctions show complementary grooves on the E face but this is extremely rare in the case of R-cell junctions. Other differences are found between R cell junctions and the "classic" continuous junction. Freeze fracture studies on continuous junctions of insect gut epithelium by Gilula (1971) and Staehelin (unpublished, see Staehelin, 1974) show a far lower number of membrane particles in the interridge areas as compared to that in our R-cell replicas. A short space (ca 10 nm) exists between adjacent aligned particles in the "usual" continuous junction (Dallai, 1975) but particles making up ridges in the R cell junction are fused with little or no intervening space. The continuous junction is a characteristic of epithelia that are continuously regenerating (Noirot and Noirot-Timoth6e, 1967). R-cells are not regenerating in that sense although protein transport and various metabolic kinetics are probably brisk in the peripheral retina. We conclude that the R-cell junction is probably a kind of continuous junction but with unique characteristics. Similarities between R cell junctions and tight junctions are obvious in freeze fractured preparations. In both cases the multiple ridges formed from aligned membrane particles make polygonal enclosures although those from tight junctions are most often completely enclosed while those from the R cell junction are often, but not always, looser in construction. The two types also differ in that membrane particles of insect tight junctions are on the E face rather than the P face as in the R cell junctions. Tight and R cell junctions are very dissimilar in thin sections. The quintuple-layered, true tight junction bears no resemblence to the two trilaminar leaflets sandwiching an intermembranous area containing indistinct septae. We are at a loss to associate with a known junction the condition in which membrane particles form short straight ridges on the P face of freeze fracture preparations without continuity or orientation (Figs. 12, 16). It is not known whether this intramembranous formation is adaptational or whether it is a function of age pertaining either to a newly forming or to a deteriorating R-cell junction. Little definite is known about the function of continuous junctions, although tight junctions have long been recognized as barriers restricting substances from

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passing through the intercellular area. Vertically oriented ridges as those in the Rcell junction could restrict horizontal diffusion currents through the intercellular space, perhaps leading to appreciable local concentration gradients. Mitochondria with their long axes parallel to, and within 10 nm of the plasma membrane, could provide energy for active transport through intercellular spaces. If, as Staehelin (1974) suggests the continuous junction is a variant of the septate junction, then cellular adhesion is likely imparted by the R-cell junction. Retinular cells of each fly ommatidium bond to their neighbors via belt desmosomes but Chi (1976), working with Musca, and Horridge, et al. (1975), studying Eristalis (also a dipteran), found that the entire fly retinula twists along its long (optical) axis. Such twisting must have a more adverse effect on E-vector discrimination by peripheral cells than on the shorter (in rhabdomeric length) central cells. R-cell junctions could provide the cohesive qualities necessary to maintain the twist. From what is known about the optics of the neural superposition eye of the fly it is unlikely that any electrical coupling exists in the peripheral retina although this phenomenon is suspected in the R-axons at the level of the lamina ganglionaris (Chi and Carlson, 1976b; Laughlin, 1975). Lateral inhibition has been found (Zettler and J/irvilehto, 1972) at this lower level but its origin may be in the peripheral retina. Locust retinular cells are circumferentially sealed so that extracellular space around the R axon does not communicate with that of the soma (Shaw, 1975, 1977). This barrier causes less stimulated R cells to hyperpolarize and, in effect, sets up lateral inhibition between the L-interneurons of the lamina. The presence of numerous membrane-aligned mitochondria implies that R-cell junctions provide more than a structural advantage. The observation that mitochondria cling to R-cell borders is not new; Gribakin (1975) comments on the functional significance of this juxtaposition in the honeybee retina. Electron micrographs (Skrzipek and Skrzipek, 1973; Menzel and Synder, 1974) show the intimate association between mitochondria and the R-cell membrane of honey bees (but no membrane specializations were discovered in that area). We do not feel that this mitochondrion-unit membrane association is influenced by fixation although Boschek (1971) mentions this as a possibility. Just as the R-cell membrane is continually changing with regard to the localization of its visual pigment and other protein molecules, the mitochondria may not maintain a tight holding pattern near the plasma membrane. In the locust, mitochondria are known to be photokinetic, a finding that has both optical and metabolic implications (Horridge and Bernhard, 1965). Dark and light adaptational studies are planned that should confirm or deny mitochondrial motility in the fly. In summary, R-cell junctions are always closely associated with mitochondria. There is experimental evidence that the fly retinula is slightly twisted and maintains a high metabolic activity. The hypothesis is offered that the R-cell junction assists both in R-cell twisting and in maintaining localized concentration gradients between R cells. Note Added in Proof

After submission of the present paper, Schinz(1978, Soc. for Neurosci. Absts. 8th Ann. meeting,St. Louis, MO, Nov. 5-9. No. 776,p. 247)brieflyreportedon a desmosomebetweenretinularcellsin which the P face"... showeda rather loose and irregular pattern of ridges, which ... displayeda particulate substraeture." No correlates of these structures were found in thin sectionsnor was any physiological role attributed to this desmosome.

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