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Ciliogenesis in photoreceptor cells of the retina
Exp. Eye Res. (t981) 33, 433-446
Ciliogenesis in Photoreceptor Cells of the Retina J A C K V. G R E I N E R * t w , T H O M A S A. W E I D M A N ~ ,
H. DANIEL
BODLEY$
AND
C Y N T H I A A. M. G R E I N E R I I
* Eye Research Institute of Retina Foundation, Boston, U.S.A. t Department of Pathology Department of Anatomy, Chicago College of Osteopathic Medicine, Chicago, U.S.A. wDepartment of Ophthalmology, University of Illinois Eye and Ear Infirmary, Chicago, U.S.A. 82Department of Anatomy, Medical College of Georgia, Augusta, Georgia, U.S.A. II Committee on Neurobiology, University of Chicago, Chicago, U.S.A. (Received 14 November 1980 and accepted 30 January 1981, New York) Ciliogenesis in the retinal photoreceptor of fetal, neonatal and adult ferrets has been investigated by electron microscopy~ Ciliogenesis is described from the time (prenatal day 21) when a diplosome (two centrioles) is observed migrating toward the region of the external limiting membrane prior to eventual alignment beneath the apical plasma membrane of photoreceptor ceils (approximately prenatal day 21). One of the centrioles becomes aligned perpendicular to the apical plasma membrane and is designated the basal body. Each developing basal body (composed of nine sets of triplet microtubules) has a rootlet extending basally and a cilium developing apically. Accessory structures associated with the basal body include foot processes and arm-like fibers. One arm-like fiber appears to be associated with each set of triplet microtubules. The arm-like fibers appear to attach to the plasma membrane along the cell apex, and it is at this point of attachment that the cell membrane invaginates. Invagination proceeds as the doublet microtubules of the developing cilium extend from the basal body toward the pigment epithelium. The invagination of the plasma membrane appears in cross-section as a ' moat' surrounding the evagination of the plasma membrane occupied by the developing cilium. This evagination containing the cilium eventually extends distal to the plane of the original photoreceptor cell apex. The developing cilium in the photoreceptor is unique in that it must overcome the resistance encountered from the adjacent pigment epithelial cell layer and does not merely grow into a lumen as in many other tissues. At maturation the invagination of the cell membrane around the cilium disappears, although the arm-like fibers are sometimes retained. Key word8: ciliogenesis; photoreceptor cells; retina; transmission electron microscopy.
1. I n t r o d u c t i o n
Cilia and their accompanying basal bodies are commonly found in the epithelia of the respiratory tract, specific parts of the male and female reproductive tracts, the ependymal cells lining the ventricles of the brain and sensory epithelial cells including the rod and cone photoreceptor cells of the retina. The process of ciliogenesis in mammalian epithelial cells has been analyzed using electron microscopy, most recently by Staprans and Dirksen (1974), Philipp (1977), and Komatsu and Fujita (1978a, b). Most studies of ciliogenesis dealt with multiciliated epithelial cells; however, Sorokin (1968) and Komatsu and Fujita (1978b) describe ciliogenesis in epithelial cells with a single cilium, similar to that found in the retinal photoreceptor cell. Developing cilia generally project into a body cavity or lumen and thus their growth is not impeded by abutting on an apposing epithelial cell surface. However, in the Reprint requests to: Thomas A. Weidman, Ph.D., Department of Anatomy, Medical College of Georgia, Augusta, Georgia 30912, U.S.A. 0014-4835/81/100433+ 14 $01.00/0
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retina the cilia grow outward toward the closely opposed pigment epithelium. Growth of the cilium occurs into a potential space (the remnant of the cavity of the optic vesicle) between the outer (pigment epithelium) and inner (sensory) walls of the optic cup and thus precedes the formation of the outer segment and the development of a functional photoreceptor cell (Greiner and Weidman, 1981). The retina of the ferret provides an excellent model for developmental studies since it develops slowly, and thus facilitates the investigation of maturing cilia (Greiner and Weidman, 1981). A partial discussion of photoreceptor ciliogenesis has been presented by other investigators (DeRobertis, 1956; Tokuyasu and Yamada, 1959) and in our previous studies (Weidman and Kuwabara, 1968; Greiner and Weidman, 1978, 1980), but no one has published a detailed account of ciliogenesis. This study examines in detail the development of the basal body complex, including the ciliary rootlet, the basal body and the connecting cilium proper in photoreeeptor cells of the ferret. 2. M e t h o d s Retinal tissue was obtained from embryos at 3-day intervals from 21 to 42 days' gestation from time-mated pregnant female ferrets, from neonates daily on postnatal days 1-30 and from adults. A minimum of three embryos from each prenatal time period and two animals from each postnatal day were examined. Pregnant females were killed by ctherization followed by cardiac puncture and their embryos were subsequently removed. Immediately following decapitation of embryos, neonates and adults, eyes were enucleated and hemisected coronally. The posterior half of each eye was fixed in cold 4 % glutaraldehyde in 0'2 M-sodium phosphate buffer, pH 7'2, for 30 min. The retina was separated from the sclera during the fixation period. The choriocapillaris was left attached to the retina. Samples of the retina obtained from the posterior pole of the eye were trimmed to a maximum dimension of 1'0 ram. Samples were transferred without washing into cold 1% osmium tetroxide in the same buffer solution and fixed for 90 min, dehydrated in graded ethyl alcohols, treated with propylene oxide and embedded in Epon. Thin sections (40-70 nm) were cut on an LKB Ultramicrotome I I I with a diamond knife, mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips 400 transmission electron microscope operated at 90 kV. 3. R e s u l t s As early as prenatal day 21 (E 21) the sensory retina has a few photoreceptors that contain paired centrioles (a diplosome) in their apical cytoplasm [Fig. 1 (a)-(c)]. Early in development every photoreceptor is found to contain a diplosome. The diplosome is initially located proximal to the external limiting membrane [Fig. 1 (a)]. The two centrioles of the diplosome sometimes are unequal in length but are considered by us to be fully grown when their length is approximately twice their diameter. One centriole of the diplosome differentiates into the basal body and the other into the associated centriole of the basal body complex. Even at early stages of development the basal body centriole can be identified by evolving satellite structures: an electron-dense basal plate and lateral foot processes [Fig. 1 (a)]. As development proceeds the diplosome displaces toward the photoreeeptor cell apex, past the external limiting membrane [Fig. 1 (b)], and eventually approaches the plasma membrane [Fig. 1 (e)]. The basal plate is at first randomly oriented during the displacement process and later becomes oriented parallel to the apical plasmalemma [Fig. 1 (c)]. The lateral foot processes extend outward approximately 0"14/xm and are perpendicular to the basal body [Fig. 1 (a), (e)]. More than one foot process per cell
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FIG, I. Prenatal retina day 21 (E 21), (a) The diplosome is located basal to the external limiting membrane (horizontal arrows). The centriole oriented in oblique section has a foot process (fp) extending laterally. Arm-like fibers at one end of the basal body project outward and connect to a basilar plate (arrowhead) (line bar = 1 ~m). (b) The diplosome is at the same level as the external limiting membrane (horizontal arrows). A basilar plate is oriented toward the apical surface (arrowhead) connected to the basal body by arm-like fibers (line bar = 1 #m). (c) The diplosome is located apically to the external limiting membrane (horizontal arrow) and has arrived at the plasmalemma. Nine sets of triplet microtubules are seen in the cross-section of the centriole. Lateral foot processes (fp) are seen on the obliquely sectioned basal body. Radiating arm-like fibers extend from the terminal end of the basal body to the plasmalemma (arrowheads). Cross-sectioned microtubules (oblique arrows) (line bar = 1 #m).
m a y be o b s e r v e d in a single p l a n e of section. The foot process is more or less cone-shaped with a b l u n t e d t i p (Fig. 2). The process a p p e a r s electron dense a t low p o w e r b u t a t higher p o w e r is f o u n d to possess a b a n d i n g p a t t e r n composed of t h r e e dense a n d two t r a n s l u c e n t bands. 'The foot process has a base d i a m e t e r of 0"06 #m. T h r o u g h o u t t h e process of d i p l o s o m e d i s p l a c e m e n t m i c r o t u b u l e s are v e r y often a s s o c i a t e d w i t h t h e d i p l o s o m a l complex [Fig. 3 (a)]. M i c r o t u b u l e s were n o t a p p a r e n t in all t h e m i c r o g r a p h s b u t when p r e s e n t t h e y were u s u a l l y i r r e g u l a r l y o r i e n t e d a n d a p p e a r to be a t t a c h e d or a s s o c i a t e d w i t h t h e centriole [Fig. 1 (a)]. I r r e g u l a r l y a r r a n g e d m i c r o t u b u l e s continue to be associated with t h e b a s a l b o d y even after the cilium matures. A ciliary r o o t l e t is a t t h e p r o x i m a l end of t h e b a s a l b o d y . This ciliary r o o t l e t m a y form as e a r l y as E 21 [Fig. 3 (b)] a n d e x t e n d s deep into the c y t o p l a s m of t h e d e v e l o p i n g inner segment. The r o o t l e t consists of l o n g i t u d i n a l l y a r r a n g e d bundles o f m i c r o f i l a m e n t s t h a t i n t e r d i g i t a t e with t r a n s v e r s e l y disposed b a n d s of electron-dense material. This b a n d i n g p a t t e r n shows a p e r i o d i c i t y of 0"06 #m. W h e n t h e displosome reaches t h e a p e x of t h e cell b o d y , one centriole, now
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FIG. 2. Prenatal retina day 21 (E 21). (a) Centriole with laterally radiating foot processes (fp). Numerous vesicles are present (line bar = 0-1/tin). (b) Longitudinal section of the basal body with foot processes (fp) showing dark and light banding. Vacuolated cytoplasm (arrows) is seen at the end of the basal body where the microtubules of the cilium are developing (line bar = 0"l #m).
FIo. 3. Prenatal retina day 21 (E 21). (a) The centriole has associated microtubules (arrows) (line bar = 0"1/~m). (b) Both centrioles of the diplosome are present, with numerous associated microtubules. The basal body has a developing striated rootlet (arrows) (line bar = 0"l #m).
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designated as the basal body, becomes oriented longitudinally (perpendicular to the plasma membrane) [Fig. 1 (c)]. The process of development of the basal body complex (i.e. ciliary rootlet, basal body, and connecting cilium) is not synchronized among cells (Fig. 4). Thus, during the course of development of the basal body complex there is variation in the timing of events among neighboring photoreceptor cells. The initiation of ciliogenesis in each cell appears to be dependent on the maturity of the individual cell, thus implying t h a t photoreceptor development in a localized region is asynchronous. A difference in cell m a t u r i t y is inherent in a difference in the birthdays of both rod and cone photoreceptors.
Fia, 4. Prenatal retina day 27 (E 27). (a) Survey electron micrograph of the junction between the apices of the pigment epithelium and developing photoreceptor cells showing different stages of ciliogenesis among neighboring cells. A basal body in cross-section has radiating arm-like fibers (vertical arrow). A developing cilium projects horizontally from a longitudinally oriented basal body (horizontal arrow) with a developing striated rootlet and a closely associated centriole (line bar = 1 #m).
In cross-section the basal body is approximately 0"1 tim in internal diameter and 0"26 ttm in external diameter and consists of nine sets of three mierotubules (triplets) in accordance with the classical description. The triplet microtubules are arranged equidistantly from the center and from each other, to form the incomplete wall of a cylinder. Each triplet is oriented parallel to the long axis of the basal body and the triplet microtubules have a clear center and a dense wall. When cut in cross- or oblique section the basal body appears as a cylinder; in longitudinal section the basal body consists of two electron-dense bands approximately 0"4 # m in length (Fig. 2). These longitudinal sections show t h a t the microtubules are straight and untwisted. The microtubules comprising the basal body and centriole are surrounded by cytoplasm that is generally more electron dense than cytoplasm found elsewhere in the cell (Figs 1, 3 and 5). Within this electron-dense region were small vesicles (0"02 #m in diameter) believed to be cross-sections ofmierotubules [Fig. 1 (e)]. This electron-dense region was relatively free of ribosomes.
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FIG. 5. Prenatal retina day 27 (E 27). (a) Survey electron micrograph of junction between apices of pigment epithelium and photoreceptor cells showing developing cilium and basal body (vertical arrows) in longitudinal section. External limiting membrane (horizontal arrows) (line bar = 1 #m). (Inset) High magnification of basal body with developing cilium. Note arm-like fibers (arrowheads) (line bar = 0'5/zm). (b) A connecting cilium extending from a basal body projecting into an invagination of the cell body. Parts of the arm=like fibers are visible (arrowheads). A vesicle is observed joining the plasmalemma (arrow) (line bar = 0-1 #m). V a c u o l a t e d c y t o p l a s m is sometimes seen near the basal p l a t e which is located at the distal end of the basal b o d y [Fig. 2 (b)]. I n oblique or t a n g e n t i a l section the basal plate a p p e a r s as a cap of electron-dense m a t e r i a l t h a t e x t e n d s down b o t h sides of the basal body. This p l a t e is located j u s t below t h e v a c u o l a t e d c y t o p l a s m and is connected to the distal end of the basal b o d y b y arm-like fibers. These arm-like fibers, which seem to arise n e a r the distal end of each t r i p l e t (Fig. 5), m a k e an angle of between 30 ~ a n d 60 ~ with the l o n g i t u d i n a l axis of the m i c r o t u b u l a r triplets a n d are oriented t o w a r d t h e apical surface of the cell. The arm-like fibers are a p p r o x i m a t e l y the same width as a single m i c r o t u b u l c of the basal body. The arm-like fibers a p p e a r to c o n t a c t the p l a s m a l e m m a [Fig. 5 (b)]. I t is a t this p o i n t of ' a t t a c h m e n t ' t h a t i n v a g i n a t i o n begins. As i n v a g i n a t i o n of the p l a s m a l e m m a proceeds, there is a simultaneous g r o w t h of m i c r o t u b u l e s from the distal end of the basal body, resulting in the d e v e l o p m e n t of the connecting cilium (Fig. 5).
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The plasma m e m b r a n e surrounds the elongating connecting cilium at all times during development. Thus, the plasma m e m b r a n e m d s t also be a u g m e n t e d during development of the cilium. A t the same time the plasma m e m b r a n e expands due to the lengthening of the ciliary microtubules and numerous vesicles (0"1 # m in diameter) are seen in the cytoplasm [Figs 1 (c) and 2 (a)]. The internal surface of these vesicles has a fuzzy coat. Some of the fuzzy vesicles appear to fuse with the plasma m e m b r a n e and are t h o u g h t to provide additional m e m b r a n e material to the lengthening plasmalemma [Fig. 5 (b)]. The connecting cilium is a modified cilium having a ring of nine paired microtubules (doublets) arranged in a circular fashion but lacking a central pair of microtubules characteristic of motile cilia (Cohen, 1969). The microtubules comprising each of the nine doublets are continuous with two of the three microtubules comprising the triplets in the basal body. The cilium develops b y elongation of the doublet microtubules, while the plasma m e m b r a n e at its base continues to invaginate (Fig. 5). Eventually, the elongating connecting cilium extends distal to the original invagination of the cell b o d y [Fig. 6 (a)]. I n cross-section this invagination is seen to encircle
Fro. 6. Prenatal retina day 36 (E 36). (a) Connecting cilium developing outward into an invagination of the cell body and outward into the extracellular space. Radiating arm-like fibers project to an electron-dense basilar plate (arrow). Numerous microtubules project toward the area of the basal body (line bar = 0"1 #m). (b) Oblique section of a cilium developing into an invagination of the cell body. In this section the associated centriole (upper right) but not the basal body of the diplosome is present (line bar = 0"1/~m). (c) Cross-section of a cilium completely surrounded by an invagination into the cell body. Plasmalemma surrounds the cilium (line bar = 0'l #m).
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FIG. 7. Prenatal retina day 36 (E 36). (a) Survey electron micrograph of basal body complex showing the basal body with foot process (fp), associated eentriole, striated rootlet (arrow), and connecting cilium. Arm-like fibers are present (white arrowheads). The invagination of the cell body is beginning to disappear (note plasmalemma, black on white arrowheads) (line bar = 1/tm). (b) Obliquely sectioned basal body. Little invagination remains (note plasmalemma, arrowheads) but there is some arm-like fiber-relatedelectron density (arrows) (line bar = 0"1 #m). (c) Basal body with a developing cilium. There is no invagination of the cell body (arrowheads). A foot process is present on the basal body. Note electron density of the plasmalemma adjacent to the cilium (line bar = 0-1 #m).
the entire developing connecting cilium, creating a m o a t - l i k e a p p e a r a n c e [Fig. 6 (b) a n d (c)]. The p l a s m a l e m m a lining the i n v a g i n a t i o n of the cell b o d y a n d s u r r o u n d i n g the connecting cilium has a fuzzy o u t e r l a y e r [Figs 5 (b) and 6 (a)] a n d therefore it is i n t e r p r e t e d as being continuous with the e x t r a c e l l u l a r space. E a r l y in d e v e l o p m e n t the elongating connecting cilium e x t e n d s obliquely t o w a r d the p i g m e n t e d epithelium a t an angle of less t h a n 90 ~ to the l o n g i t u d i n a l axis of the cell [Fig. 7 (a)]. As d e v e l o p m e n t proceeds, there is further lengthening of t h e cilium a n d the angle decreases until the connecting cilium is oriented a p p r o x i m a t e l y parallel with the basal body, ciliary rootlet a n d the cell [Fig. 7 (b) a n d (c)]. The p l a s m a m e m b r a n e s u r r o u n d i n g the connecting cilium has grown o u t w a r d t o w a r d the p i g m e n t e d
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(c) FIG. 8. Postnatal retina day 30 (P 30). (a) The basal body complex (bottom). Basal portion of the photoreceptor is represented schematically (top). At bottom of micrograph the developing outer segment is seen with a stack of early developing membranous discs. Proceeding upward is the connecting cilium, which joins the developing outer segment with the inner segment. The basal body (arrowhead) from which the connecting cilium develops is seen. The associated centriole is above and to the left of the basal body. The striated rootlet extends upward into the inner segment. External limiting membrane (arrows). The schematic shows the nuclear and synaptic regions (line bar = l #m). (b) Adult retina. High magnification of basal body complex with basal body and associated ciliary rootlet and connecting cilium. The basal body has a lateral foot process (fp) with associated microtubules in both longitudinaland cross-sectional profiles. Arm-like fibers slant outward from the lower two-thirds of the basal body and contact the plasmalemma (arrowheads). Striated rootlet with two bundles of microfilaments. The inner bundle (arrow) is associated with the nearby centriole (line bar = 0'1 #m). (c) Schematic representation of the basal body complex. e p i t h e l i u m [Fig. 7 (c)] a n d the ' m o a t ' disappears. The p l a s m a m e m b r a n e at the distal end of the connecting cilium will e v e n t u a l l y balloon, a n d the first disc m e m b r a n e s of the developing outer s e g m e n t will form from it (Fig. 8) ( W e i d m a n a n d K u w a b a r a , 1968; Greiner a n d W e i d m a n , 1978). The basal b o d y complex, consisting of the basal body proper, the associated centriole, the ciliary rootlet a n d the c o n n e c t i n g cilium, is located eccentrically in' the cell b o d y (Fig. 8). The ciliary rootlet appears to consist
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of two bundles of microfilaments that originate from the proximal end of the basal body (Fig. 8). One of the microfilamentous bundles appears to originate from the most peripherally (or laterally) located triplet of the basal body. In these specimens this bundle is more extensive than the second bundle that originates from the most medially located triplet. This medially originating microfilament bundle extends only a short distance and is frequently found related to the associated centriole. Both groups of microfilaments descend within the cell more or less parallel to the axis of the basal body and the cell. In the mature basal body complex, the ciliary rootlet was longer than the connecting cilium. However, serial sectioning was not performed to determine the exact length. Membrane discs forming the outer segment develop from the distal end of the connecting cilium. The cilium becomes located in the peripheral cytoplasm of the developing outer segment [Fig. 8 (b)]. Microtubules extend beyond the connecting cilium proper into the developing outer segment, but not as an organized cilium. The foot process that extends laterally from the basal body and the arm-like fibers are still present. The arm-like fibers appear to remain attached to the plasma membrane. 4. D i s c u s s i o n
Retinal ciliogenesis The development of the basal body complex is an integral part of the process of eiliogenesis. The two centrioles composing the diplosome migrate toward the apical surface of the developing photoreceptor cells. Microtubules are commonly observed with the diplosome. I t is possible that these microtubules participate in the relocation and reorientation of the diplosome and in its final location at the cell membrane. Microtubules are always associated with centrioles during spindle formation in a dividing cell. Thus their association with the diplosome in the developing cilium is not surprising. Therefore, the basal body in the photoreceptor inner segment may function in the production of microtubules for the cilium, as does the centriole in spindle tubule formation. One centriole differentiates into a basal body while the other remains as the associated centriole. An electron-dense projection, basal foot, extends laterally from the mideentral region of the basal body during relocation distad. This basal foot process has also been referred to as a satellite arm (Sorokin, 1968) or fibrous tuft (Cohen, 1969). The number of foot processes per basal body is not known: however, our findings indicate that there are at least three in each photoreceptor cell in the retina. The banding pattern characteristic of the foot process was first described by deHarven and Bernhard (1956) in the respiratory tissue. Prior to its arrival at the plasmalemma, arm-like fibers form from one end of the basal body. These fibers appear to join to an electron-dense area. known as the basal plate [Fig. 9 (a)-(c)]. The vacuoles associated with the basal plate are similar to cytoplasmic vesicles described by Sorokin (1968). Sorokin suggested that these vesicles originate in the ctyoplasm as a result of an interaction between the basal body and the Golgi lamellae; the vacuoles are then thought to fuse with the plasmalemma. Upon arrival at the apex of the cell the basal plate seems to join with the plasma membrane, and the arm-like fibers appear to 'pull' the plasmalemma at the points of contact [Fig. 9 (d)-(f)]. The arm-like fibers that extend from the distal (apical) end of the basal body have not been described. Serial sectioning being done for a separate study may reveal the
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FIO. 9. Schematic representation of a proposed model of ciliogenesis in the retinal photoreceptor presented sequentially from left to right. (a) The diplosome resides in the apical cytoplasm ; one centriole (basal body) is attached to a basilar plate by arm-like fibers. The basilar plate borders an area of vaeuolated cytoplasm. (b) The basal body and basilar plate become closely associated with the plasmalemma. (c) The arm-like fibers 'tug' on the plasmalemma, initiating invagination. (d) The plasmalemma begins to invaginate, as the microtubules of the cilium begin to develop. (e)-(g) Invagination of the plasmalemma and lengthening of the cilium continues. (h) Oblique angle of developing cilium and the onset of disc formation. (i) Matured cilium with developing stack of disc membranes. precise m o r p h o l o g y of this structure. The arm-like fiber associated with the apex of the cell m a y create tension on the p l a s m a l e m m a resulting in an invagination of the cell m e m b r a n e [Fig. 9 (d)-(f)]. I t is i m p o r t a n t to remember t h a t each triplet of the basal b o d y has an arm-like fiber and thus invagination occurs in a ringlike fashion to produce a ' m o a t - l i k e ' appearance in appropriate cross-sections (Fig. 7). A similar form of invagination of the cell m e m b r a n e has been described in developing cilia of embryonic cartilage (Scherft and Daems, 1967), fetal rat lung (Sorokin, 1968), primordial upper respiratory tract (Friedmann and Bird, 1971), uterine endocervix (Philipp, 1977) and articular cartilage (Meier-Vismara, Walker and Vogel, 1979). At the same time t h a t the p l a s m a l e m m a invaginates at the apex of the cell body, doublet microtubules from each triplet elongate to form an evagination of the plasmalemma
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or ciliary bud. Further elongation of the ciliary bud results in the formation of the connecting cilium [Fig. 9 (g)-(i)]. The striated rootlet (ciliary rootlet) in ferret photoreceptors develops prior to or simultaneous with the invagination process and the development of the ciliary bud. In another study, Ash and Stephens (1975) reported that fully elongated cilia were present in molluscan gill filaments with only ' some indication' of the ciliary rootlets. This suggests that the ciliary rootlet grew late in the formation of the cilium in these specimens. The difference between our findings and theirs may be due to a species variation, variability in the plane of sectioning, or a high degree of variability from cell to cell. The striated rootlet in the mammalian photoreceptor cell may provide support for the basal body. Sebuwufu (1968) suggests that the main function of the rootlet is to anchor the cilium in the cell. The arm-like fibers described in this study may serve both supportive and anchoring functions by stabilizing the basal body. These arm-like fibers may be related to the anchoring system described by Sorokin (1962) in the fetal rat lung. Mattern, Daniel and Henkin (1970) reported nine radiating spokes in the ciliated cells of the tongue which may also be related to the arm-like fibers described in our study. Anderson (1972) observed fibers in the rhesus monkey oviduct that originate from the foot processes to reach the plasmalemma. An anchoring system binding the peripheral part of the basal body to the plasmalemma was described by Cordier (1975). Microtubules existing in a random fashion around the basal body may also contribute to supportive and anchoring functions. The developing cilium is covered by plasma membrane; therefore, as the cilium lengthens, there must be a concomitant increase in the plasma membrane. Our observations suggest that cytoplasmic vesicles fuse with the apical plasma membrane, thus resulting in an increase of the membrane. A similar phenomenon was suggested by Steinman (1968) and Komatsu and Fujita (1978a). This is supported by our observation that these smooth vesicles can be found fused with the plasma membrane in the region of the cilium; however, the direction of movement of these vesicles cannot be determined by electron micrographs alone. As the cilium lengthens, its distal end eventually projects past the apex of the cell [Fig. 9 (f) and (g)]. At first, only a few cilia extend into the potential space between the outer layer ofphotoreceptor cells and the pigment epithelium. The developing cilia abut the apposing pigmented epithelium. As described here and in our previous studies of the mammalian retina (Weidman and Kuwabara, 1968; Greiner and Weidman, 1978, 1980), the developing cilium initially grows at an angle; it later straightens to become oriented parallel to the long axis of the cell. We believe that the opposing force presented by the pigmented epithelium causes the cilium to develop initially at an angle to the longitudinal axis of the photoreceptor cell. However, as cilium development proceeds and becomes more or less synchronous, the force exerted by the growth and straightening of a large number of cilia increases the distance between the pigment epithelium and the photoreceptor cell bodies, allowing initial straight growth of later-developing cilia. The morphologie evidence presented herein supports the hypothesis that ciliogenesis is complete when disc membranes form at the distal end of the cilium, resulting in the development of the outer segment.
Comparative ciliogenesis Unlike most ciliated cells, which have multiple motile cilia, the photoreceptor cell of the retina has a single nonmotile cilium. The cell with multiple cilia has a solitary
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centriole (basal body) associated with each cilium. Single (primary) cilia have a diplosome consisting of one basal body and of one associated centriole. In cross-section the single cilium lacks the central pair of ciliary fibers (a 9 + 0 microtubular pattern) common to a motile cilium. Single cilia occur in sensory cells such as retinal photoreceptors and vestibular hair cells as well as in a number of undifferentiated cells which are nonciliated when mature. The transitory existence of the latter has been described by Sorokiu (1968). Cells with a single cilium have been reported in mammalian tissues such as connective tissue (Meier-Vismara et al., 1979), glandular tissue (Kamatsu and Fujita, 1978b), muscle (Przybylski, 1971), kidney (Webber and Lee, !975), and nervous tissue (del Carro and Snider, 1969}. Frequently these cilia are found in the cytoplasm or in vacuoles or invagiuatious of the plasmalemma, the nuclear membrane and elsewhere (Friedmann and Bird, 1971). Scherft and DaemS (1967) suggest ' t h a t the potency to form single cilia is present in almost every kind of tissue', but the significance of most of these cilia is still obscure. For example, in describing the single cilium of the articular cartilage of the cat, Meier-Vismara et al. (1979) observed the majority of the single cilia undergoing involution. Although in most cells solitary cilia are transitory and tend to decrease in number with maturation, these structures sometimes persist in the fully differentiated cells, e.g. secretory cells (Komatsu and Fuj!ta, 1978b) and sensory cells such as the retinal photoreceptor, in the retinal photoreceptor the early embryonic cilium does not involute but serves as the connecting piece between the inner and outer segments. Ciliary rootlets are present in both singly and multiply ciliated cells. However, the shape of these rootlets varies among cell types. In contrast to the multiciliated cell, the ciliary rootlets of the photoreceptor cells and other singly ciliated cells do not diverge as widely as they extend basally in the cell cytoplasm. Solitary cilia in many epithelia undergo a similar developmental process as in the retinal photoreceptor cilium. In fully developed retinal photoreceptors, the connecting cilia are uniform in length. Sorokin's (1968) observation that primary cilia of the epithelial border of the lung are of variable length may be an artifact of sectioning, since these cilia are randomly oriented. Thus it appears that the 'primary cilium' of retinal photoreceptors does fall into the rudimentary category developmentally but instead of involuting the primary cilium remains throughout life and serves to connect the inner and outer segments of the photor~eeptor. This has great significance for what is known concerning disc membrane production and synthesis, and transport of biologically active components of the photoreceptor membrane. ACKNOWLEDGMENTS This study was supported by Institutional National Research Service Award EY-07018 awarded to the Eye Research Institute and by core grant EY-01792 to the Department of Ophthalmology, University of Illinois by the National Eye Institute, National Institutes of Health. The authors thank Ms Ruth Zelkha for photography and Ms Bettye Harris and Mrs Marjorie Pole for the typing of the manuscript. REFERENCES Anderson, R. G. W. (1972). The three dimensional structure of the basal body from the rhesus monkey oviduct. J. Cell Biol. 54, 246-65. Ash, B.M. and Stephens, R.E. (1975). Ciliogenesis during the sequential formation of Molluscan gill filaments. Dev. Biol. 43, 340-7.
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del Cerro, M. P. and Snider, R. S. (1969). The Purkinje cell cilium. Anat. Rec. 165, 127-40. Cohen, A. I. (1969). Rods and cones and the problem of visual excitation. In The Retina. Morphology, Function and Clinical Characteristics (Eds Straatsma, B. R., Hall, M. 0., Allen, R. A. and Creseitelli, F.). Pp. 31-72. University of California Press, Berkeley and Los Angeles. Cordier, A. C. (1975). Ciliogenesis and ciliary anomalies in thymic cysts o f ' n u d e ' mice. Cell Tissue Res. 148, 397-406. DeRobertis, E. (1956). Morphogenesis of the retinal rods. J. Biophys. Biochem. Cytol. 2 (Supp.), 209-18. Friedmann, I. and Bird, E. S. (1971). Ciliary structure, ciliogenesis, microvilli. Electron" microscopy of the mucosa of the upper respiratory tract. Laryngoscope 81, 1852-68. Greiner, J. V. and Weidman, T. A. (1978). Development of the hamster retina. A morphologic study. Am. J. Vet. Res. 39, 665-70. Greiner, J. V. and Weidmann, T. A. (1980). Histogenesis of the eat retina. Exp. Eye Res. 30, 439-53. Greiner, J. V. and Weidmann, T. A. (1981). Histogenesis of the ferret retina. Exp. Eye Res. 33, 315-32. deHarven, E. and Bernhard, W. (1956). Etude au microscope 61ectronique de 1'ultrastructure du centriole chez les vert6br6s. Z. Zellforsch. Mikrosk. Anat. 45,378-98. Komatsu, M. and Fujita, H. (1978a). Some observations on eiliogenesis in the oviduct epithelium of the mouse. Arch. Histol. Jpn 41,229-37. Komatsu, M. and Fujita, H. (1978b). Electron-microscopic studies on the development and aging of the oviduct epithelium of mice. Anat. Embryol. 152, 243-59. Mattern, C. F. T., Daniel, W. A. and Henkin, R. I. (1970). The ultrastructure of the htiman circumvallate papilla. I. Cilia of the papillary crypt. Anat. Rec. I67, 175-82. Meier-Vismara, E., Walker, N. and Vogel, A. (1979). Single cilia in the articular cartilage of the cat. Exp. Cell Biol. 47, 161-71. Philipp, E. (1977). Elektronenmikroskopische Untersuchiangen fiber die Ciliogenese in Flimmerzellen der menschliehen Endocervix uteri. Med. Welt 28, 563-68. Przybylski, R. J. (1971). Occurence of centrioles during skeletal and cardiac myogenesis. J. Cell Biol. 48, 214-21. Scherft, J. P. and Daems, W. T. (1967). Single cilia in chondrocytes. J. Ultrastruct. Res. 19, 546-55. Sebuwufu, P. H. (1968). Ultrastructure of human fetal thymic cilia. J. Ultrastruct. Res. 24, 171-80.
Sorokin, S. (1962). Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J. Cell Biol. 15,363-77. Sorokin, S. P. (1968). Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J. Cell Sci. 3, 207-30. Staprans, I. and Dirksen, E. R. (1974). Microtubule protein during eiliogenesis in the mouse oviduct. J. Cell Biol. 52, 164-74. Steinman, R. M. (1968). An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis. Am. J. Anat. 122, 19-56. Tokuyasu, K. and Yamada, E. (1959). The fine structure of the retina studied with the electron microscope. IV. Morphogenesis of outer segments of retinal rods. J. Biophys. Biochem. Cytol. 5, 225-30. Webber, W. A. and Lee, J. (1975). Fine structure of mammalian renal cilia. Anat. Rec. 182, 339-44. Weidman, T. A. and Kuwabara, T. (1968). Postnatal development of the rat retina. An electron microscopic study. Arch. Ophthalmol. 79, 470-84.
Ciliogenesis in Photoreceptor Cells of the Retina J A C K V. G R E I N E R * t w , T H O M A S A. W E I D M A N ~ ,
H. DANIEL
BODLEY$
AND
C Y N T H I A A. M. G R E I N E R I I
* Eye Research Institute of Retina Foundation, Boston, U.S.A. t Department of Pathology Department of Anatomy, Chicago College of Osteopathic Medicine, Chicago, U.S.A. wDepartment of Ophthalmology, University of Illinois Eye and Ear Infirmary, Chicago, U.S.A. 82Department of Anatomy, Medical College of Georgia, Augusta, Georgia, U.S.A. II Committee on Neurobiology, University of Chicago, Chicago, U.S.A. (Received 14 November 1980 and accepted 30 January 1981, New York) Ciliogenesis in the retinal photoreceptor of fetal, neonatal and adult ferrets has been investigated by electron microscopy~ Ciliogenesis is described from the time (prenatal day 21) when a diplosome (two centrioles) is observed migrating toward the region of the external limiting membrane prior to eventual alignment beneath the apical plasma membrane of photoreceptor ceils (approximately prenatal day 21). One of the centrioles becomes aligned perpendicular to the apical plasma membrane and is designated the basal body. Each developing basal body (composed of nine sets of triplet microtubules) has a rootlet extending basally and a cilium developing apically. Accessory structures associated with the basal body include foot processes and arm-like fibers. One arm-like fiber appears to be associated with each set of triplet microtubules. The arm-like fibers appear to attach to the plasma membrane along the cell apex, and it is at this point of attachment that the cell membrane invaginates. Invagination proceeds as the doublet microtubules of the developing cilium extend from the basal body toward the pigment epithelium. The invagination of the plasma membrane appears in cross-section as a ' moat' surrounding the evagination of the plasma membrane occupied by the developing cilium. This evagination containing the cilium eventually extends distal to the plane of the original photoreceptor cell apex. The developing cilium in the photoreceptor is unique in that it must overcome the resistance encountered from the adjacent pigment epithelial cell layer and does not merely grow into a lumen as in many other tissues. At maturation the invagination of the cell membrane around the cilium disappears, although the arm-like fibers are sometimes retained. Key word8: ciliogenesis; photoreceptor cells; retina; transmission electron microscopy.
1. I n t r o d u c t i o n
Cilia and their accompanying basal bodies are commonly found in the epithelia of the respiratory tract, specific parts of the male and female reproductive tracts, the ependymal cells lining the ventricles of the brain and sensory epithelial cells including the rod and cone photoreceptor cells of the retina. The process of ciliogenesis in mammalian epithelial cells has been analyzed using electron microscopy, most recently by Staprans and Dirksen (1974), Philipp (1977), and Komatsu and Fujita (1978a, b). Most studies of ciliogenesis dealt with multiciliated epithelial cells; however, Sorokin (1968) and Komatsu and Fujita (1978b) describe ciliogenesis in epithelial cells with a single cilium, similar to that found in the retinal photoreceptor cell. Developing cilia generally project into a body cavity or lumen and thus their growth is not impeded by abutting on an apposing epithelial cell surface. However, in the Reprint requests to: Thomas A. Weidman, Ph.D., Department of Anatomy, Medical College of Georgia, Augusta, Georgia 30912, U.S.A. 0014-4835/81/100433+ 14 $01.00/0
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retina the cilia grow outward toward the closely opposed pigment epithelium. Growth of the cilium occurs into a potential space (the remnant of the cavity of the optic vesicle) between the outer (pigment epithelium) and inner (sensory) walls of the optic cup and thus precedes the formation of the outer segment and the development of a functional photoreceptor cell (Greiner and Weidman, 1981). The retina of the ferret provides an excellent model for developmental studies since it develops slowly, and thus facilitates the investigation of maturing cilia (Greiner and Weidman, 1981). A partial discussion of photoreceptor ciliogenesis has been presented by other investigators (DeRobertis, 1956; Tokuyasu and Yamada, 1959) and in our previous studies (Weidman and Kuwabara, 1968; Greiner and Weidman, 1978, 1980), but no one has published a detailed account of ciliogenesis. This study examines in detail the development of the basal body complex, including the ciliary rootlet, the basal body and the connecting cilium proper in photoreeeptor cells of the ferret. 2. M e t h o d s Retinal tissue was obtained from embryos at 3-day intervals from 21 to 42 days' gestation from time-mated pregnant female ferrets, from neonates daily on postnatal days 1-30 and from adults. A minimum of three embryos from each prenatal time period and two animals from each postnatal day were examined. Pregnant females were killed by ctherization followed by cardiac puncture and their embryos were subsequently removed. Immediately following decapitation of embryos, neonates and adults, eyes were enucleated and hemisected coronally. The posterior half of each eye was fixed in cold 4 % glutaraldehyde in 0'2 M-sodium phosphate buffer, pH 7'2, for 30 min. The retina was separated from the sclera during the fixation period. The choriocapillaris was left attached to the retina. Samples of the retina obtained from the posterior pole of the eye were trimmed to a maximum dimension of 1'0 ram. Samples were transferred without washing into cold 1% osmium tetroxide in the same buffer solution and fixed for 90 min, dehydrated in graded ethyl alcohols, treated with propylene oxide and embedded in Epon. Thin sections (40-70 nm) were cut on an LKB Ultramicrotome I I I with a diamond knife, mounted on copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips 400 transmission electron microscope operated at 90 kV. 3. R e s u l t s As early as prenatal day 21 (E 21) the sensory retina has a few photoreceptors that contain paired centrioles (a diplosome) in their apical cytoplasm [Fig. 1 (a)-(c)]. Early in development every photoreceptor is found to contain a diplosome. The diplosome is initially located proximal to the external limiting membrane [Fig. 1 (a)]. The two centrioles of the diplosome sometimes are unequal in length but are considered by us to be fully grown when their length is approximately twice their diameter. One centriole of the diplosome differentiates into the basal body and the other into the associated centriole of the basal body complex. Even at early stages of development the basal body centriole can be identified by evolving satellite structures: an electron-dense basal plate and lateral foot processes [Fig. 1 (a)]. As development proceeds the diplosome displaces toward the photoreeeptor cell apex, past the external limiting membrane [Fig. 1 (b)], and eventually approaches the plasma membrane [Fig. 1 (e)]. The basal plate is at first randomly oriented during the displacement process and later becomes oriented parallel to the apical plasmalemma [Fig. 1 (c)]. The lateral foot processes extend outward approximately 0"14/xm and are perpendicular to the basal body [Fig. 1 (a), (e)]. More than one foot process per cell
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FIG, I. Prenatal retina day 21 (E 21), (a) The diplosome is located basal to the external limiting membrane (horizontal arrows). The centriole oriented in oblique section has a foot process (fp) extending laterally. Arm-like fibers at one end of the basal body project outward and connect to a basilar plate (arrowhead) (line bar = 1 ~m). (b) The diplosome is at the same level as the external limiting membrane (horizontal arrows). A basilar plate is oriented toward the apical surface (arrowhead) connected to the basal body by arm-like fibers (line bar = 1 #m). (c) The diplosome is located apically to the external limiting membrane (horizontal arrow) and has arrived at the plasmalemma. Nine sets of triplet microtubules are seen in the cross-section of the centriole. Lateral foot processes (fp) are seen on the obliquely sectioned basal body. Radiating arm-like fibers extend from the terminal end of the basal body to the plasmalemma (arrowheads). Cross-sectioned microtubules (oblique arrows) (line bar = 1 #m).
m a y be o b s e r v e d in a single p l a n e of section. The foot process is more or less cone-shaped with a b l u n t e d t i p (Fig. 2). The process a p p e a r s electron dense a t low p o w e r b u t a t higher p o w e r is f o u n d to possess a b a n d i n g p a t t e r n composed of t h r e e dense a n d two t r a n s l u c e n t bands. 'The foot process has a base d i a m e t e r of 0"06 #m. T h r o u g h o u t t h e process of d i p l o s o m e d i s p l a c e m e n t m i c r o t u b u l e s are v e r y often a s s o c i a t e d w i t h t h e d i p l o s o m a l complex [Fig. 3 (a)]. M i c r o t u b u l e s were n o t a p p a r e n t in all t h e m i c r o g r a p h s b u t when p r e s e n t t h e y were u s u a l l y i r r e g u l a r l y o r i e n t e d a n d a p p e a r to be a t t a c h e d or a s s o c i a t e d w i t h t h e centriole [Fig. 1 (a)]. I r r e g u l a r l y a r r a n g e d m i c r o t u b u l e s continue to be associated with t h e b a s a l b o d y even after the cilium matures. A ciliary r o o t l e t is a t t h e p r o x i m a l end of t h e b a s a l b o d y . This ciliary r o o t l e t m a y form as e a r l y as E 21 [Fig. 3 (b)] a n d e x t e n d s deep into the c y t o p l a s m of t h e d e v e l o p i n g inner segment. The r o o t l e t consists of l o n g i t u d i n a l l y a r r a n g e d bundles o f m i c r o f i l a m e n t s t h a t i n t e r d i g i t a t e with t r a n s v e r s e l y disposed b a n d s of electron-dense material. This b a n d i n g p a t t e r n shows a p e r i o d i c i t y of 0"06 #m. W h e n t h e displosome reaches t h e a p e x of t h e cell b o d y , one centriole, now
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FIG. 2. Prenatal retina day 21 (E 21). (a) Centriole with laterally radiating foot processes (fp). Numerous vesicles are present (line bar = 0-1/tin). (b) Longitudinal section of the basal body with foot processes (fp) showing dark and light banding. Vacuolated cytoplasm (arrows) is seen at the end of the basal body where the microtubules of the cilium are developing (line bar = 0"l #m).
FIo. 3. Prenatal retina day 21 (E 21). (a) The centriole has associated microtubules (arrows) (line bar = 0"1/~m). (b) Both centrioles of the diplosome are present, with numerous associated microtubules. The basal body has a developing striated rootlet (arrows) (line bar = 0"l #m).
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designated as the basal body, becomes oriented longitudinally (perpendicular to the plasma membrane) [Fig. 1 (c)]. The process of development of the basal body complex (i.e. ciliary rootlet, basal body, and connecting cilium) is not synchronized among cells (Fig. 4). Thus, during the course of development of the basal body complex there is variation in the timing of events among neighboring photoreceptor cells. The initiation of ciliogenesis in each cell appears to be dependent on the maturity of the individual cell, thus implying t h a t photoreceptor development in a localized region is asynchronous. A difference in cell m a t u r i t y is inherent in a difference in the birthdays of both rod and cone photoreceptors.
Fia, 4. Prenatal retina day 27 (E 27). (a) Survey electron micrograph of the junction between the apices of the pigment epithelium and developing photoreceptor cells showing different stages of ciliogenesis among neighboring cells. A basal body in cross-section has radiating arm-like fibers (vertical arrow). A developing cilium projects horizontally from a longitudinally oriented basal body (horizontal arrow) with a developing striated rootlet and a closely associated centriole (line bar = 1 #m).
In cross-section the basal body is approximately 0"1 tim in internal diameter and 0"26 ttm in external diameter and consists of nine sets of three mierotubules (triplets) in accordance with the classical description. The triplet microtubules are arranged equidistantly from the center and from each other, to form the incomplete wall of a cylinder. Each triplet is oriented parallel to the long axis of the basal body and the triplet microtubules have a clear center and a dense wall. When cut in cross- or oblique section the basal body appears as a cylinder; in longitudinal section the basal body consists of two electron-dense bands approximately 0"4 # m in length (Fig. 2). These longitudinal sections show t h a t the microtubules are straight and untwisted. The microtubules comprising the basal body and centriole are surrounded by cytoplasm that is generally more electron dense than cytoplasm found elsewhere in the cell (Figs 1, 3 and 5). Within this electron-dense region were small vesicles (0"02 #m in diameter) believed to be cross-sections ofmierotubules [Fig. 1 (e)]. This electron-dense region was relatively free of ribosomes.
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FIG. 5. Prenatal retina day 27 (E 27). (a) Survey electron micrograph of junction between apices of pigment epithelium and photoreceptor cells showing developing cilium and basal body (vertical arrows) in longitudinal section. External limiting membrane (horizontal arrows) (line bar = 1 #m). (Inset) High magnification of basal body with developing cilium. Note arm-like fibers (arrowheads) (line bar = 0'5/zm). (b) A connecting cilium extending from a basal body projecting into an invagination of the cell body. Parts of the arm=like fibers are visible (arrowheads). A vesicle is observed joining the plasmalemma (arrow) (line bar = 0-1 #m). V a c u o l a t e d c y t o p l a s m is sometimes seen near the basal p l a t e which is located at the distal end of the basal b o d y [Fig. 2 (b)]. I n oblique or t a n g e n t i a l section the basal plate a p p e a r s as a cap of electron-dense m a t e r i a l t h a t e x t e n d s down b o t h sides of the basal body. This p l a t e is located j u s t below t h e v a c u o l a t e d c y t o p l a s m and is connected to the distal end of the basal b o d y b y arm-like fibers. These arm-like fibers, which seem to arise n e a r the distal end of each t r i p l e t (Fig. 5), m a k e an angle of between 30 ~ a n d 60 ~ with the l o n g i t u d i n a l axis of the m i c r o t u b u l a r triplets a n d are oriented t o w a r d t h e apical surface of the cell. The arm-like fibers are a p p r o x i m a t e l y the same width as a single m i c r o t u b u l c of the basal body. The arm-like fibers a p p e a r to c o n t a c t the p l a s m a l e m m a [Fig. 5 (b)]. I t is a t this p o i n t of ' a t t a c h m e n t ' t h a t i n v a g i n a t i o n begins. As i n v a g i n a t i o n of the p l a s m a l e m m a proceeds, there is a simultaneous g r o w t h of m i c r o t u b u l e s from the distal end of the basal body, resulting in the d e v e l o p m e n t of the connecting cilium (Fig. 5).
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The plasma m e m b r a n e surrounds the elongating connecting cilium at all times during development. Thus, the plasma m e m b r a n e m d s t also be a u g m e n t e d during development of the cilium. A t the same time the plasma m e m b r a n e expands due to the lengthening of the ciliary microtubules and numerous vesicles (0"1 # m in diameter) are seen in the cytoplasm [Figs 1 (c) and 2 (a)]. The internal surface of these vesicles has a fuzzy coat. Some of the fuzzy vesicles appear to fuse with the plasma m e m b r a n e and are t h o u g h t to provide additional m e m b r a n e material to the lengthening plasmalemma [Fig. 5 (b)]. The connecting cilium is a modified cilium having a ring of nine paired microtubules (doublets) arranged in a circular fashion but lacking a central pair of microtubules characteristic of motile cilia (Cohen, 1969). The microtubules comprising each of the nine doublets are continuous with two of the three microtubules comprising the triplets in the basal body. The cilium develops b y elongation of the doublet microtubules, while the plasma m e m b r a n e at its base continues to invaginate (Fig. 5). Eventually, the elongating connecting cilium extends distal to the original invagination of the cell b o d y [Fig. 6 (a)]. I n cross-section this invagination is seen to encircle
Fro. 6. Prenatal retina day 36 (E 36). (a) Connecting cilium developing outward into an invagination of the cell body and outward into the extracellular space. Radiating arm-like fibers project to an electron-dense basilar plate (arrow). Numerous microtubules project toward the area of the basal body (line bar = 0"1 #m). (b) Oblique section of a cilium developing into an invagination of the cell body. In this section the associated centriole (upper right) but not the basal body of the diplosome is present (line bar = 0"1/~m). (c) Cross-section of a cilium completely surrounded by an invagination into the cell body. Plasmalemma surrounds the cilium (line bar = 0'l #m).
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FIG. 7. Prenatal retina day 36 (E 36). (a) Survey electron micrograph of basal body complex showing the basal body with foot process (fp), associated eentriole, striated rootlet (arrow), and connecting cilium. Arm-like fibers are present (white arrowheads). The invagination of the cell body is beginning to disappear (note plasmalemma, black on white arrowheads) (line bar = 1/tm). (b) Obliquely sectioned basal body. Little invagination remains (note plasmalemma, arrowheads) but there is some arm-like fiber-relatedelectron density (arrows) (line bar = 0"1 #m). (c) Basal body with a developing cilium. There is no invagination of the cell body (arrowheads). A foot process is present on the basal body. Note electron density of the plasmalemma adjacent to the cilium (line bar = 0-1 #m).
the entire developing connecting cilium, creating a m o a t - l i k e a p p e a r a n c e [Fig. 6 (b) a n d (c)]. The p l a s m a l e m m a lining the i n v a g i n a t i o n of the cell b o d y a n d s u r r o u n d i n g the connecting cilium has a fuzzy o u t e r l a y e r [Figs 5 (b) and 6 (a)] a n d therefore it is i n t e r p r e t e d as being continuous with the e x t r a c e l l u l a r space. E a r l y in d e v e l o p m e n t the elongating connecting cilium e x t e n d s obliquely t o w a r d the p i g m e n t e d epithelium a t an angle of less t h a n 90 ~ to the l o n g i t u d i n a l axis of the cell [Fig. 7 (a)]. As d e v e l o p m e n t proceeds, there is further lengthening of t h e cilium a n d the angle decreases until the connecting cilium is oriented a p p r o x i m a t e l y parallel with the basal body, ciliary rootlet a n d the cell [Fig. 7 (b) a n d (c)]. The p l a s m a m e m b r a n e s u r r o u n d i n g the connecting cilium has grown o u t w a r d t o w a r d the p i g m e n t e d
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(c) FIG. 8. Postnatal retina day 30 (P 30). (a) The basal body complex (bottom). Basal portion of the photoreceptor is represented schematically (top). At bottom of micrograph the developing outer segment is seen with a stack of early developing membranous discs. Proceeding upward is the connecting cilium, which joins the developing outer segment with the inner segment. The basal body (arrowhead) from which the connecting cilium develops is seen. The associated centriole is above and to the left of the basal body. The striated rootlet extends upward into the inner segment. External limiting membrane (arrows). The schematic shows the nuclear and synaptic regions (line bar = l #m). (b) Adult retina. High magnification of basal body complex with basal body and associated ciliary rootlet and connecting cilium. The basal body has a lateral foot process (fp) with associated microtubules in both longitudinaland cross-sectional profiles. Arm-like fibers slant outward from the lower two-thirds of the basal body and contact the plasmalemma (arrowheads). Striated rootlet with two bundles of microfilaments. The inner bundle (arrow) is associated with the nearby centriole (line bar = 0'1 #m). (c) Schematic representation of the basal body complex. e p i t h e l i u m [Fig. 7 (c)] a n d the ' m o a t ' disappears. The p l a s m a m e m b r a n e at the distal end of the connecting cilium will e v e n t u a l l y balloon, a n d the first disc m e m b r a n e s of the developing outer s e g m e n t will form from it (Fig. 8) ( W e i d m a n a n d K u w a b a r a , 1968; Greiner a n d W e i d m a n , 1978). The basal b o d y complex, consisting of the basal body proper, the associated centriole, the ciliary rootlet a n d the c o n n e c t i n g cilium, is located eccentrically in' the cell b o d y (Fig. 8). The ciliary rootlet appears to consist
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of two bundles of microfilaments that originate from the proximal end of the basal body (Fig. 8). One of the microfilamentous bundles appears to originate from the most peripherally (or laterally) located triplet of the basal body. In these specimens this bundle is more extensive than the second bundle that originates from the most medially located triplet. This medially originating microfilament bundle extends only a short distance and is frequently found related to the associated centriole. Both groups of microfilaments descend within the cell more or less parallel to the axis of the basal body and the cell. In the mature basal body complex, the ciliary rootlet was longer than the connecting cilium. However, serial sectioning was not performed to determine the exact length. Membrane discs forming the outer segment develop from the distal end of the connecting cilium. The cilium becomes located in the peripheral cytoplasm of the developing outer segment [Fig. 8 (b)]. Microtubules extend beyond the connecting cilium proper into the developing outer segment, but not as an organized cilium. The foot process that extends laterally from the basal body and the arm-like fibers are still present. The arm-like fibers appear to remain attached to the plasma membrane. 4. D i s c u s s i o n
Retinal ciliogenesis The development of the basal body complex is an integral part of the process of eiliogenesis. The two centrioles composing the diplosome migrate toward the apical surface of the developing photoreceptor cells. Microtubules are commonly observed with the diplosome. I t is possible that these microtubules participate in the relocation and reorientation of the diplosome and in its final location at the cell membrane. Microtubules are always associated with centrioles during spindle formation in a dividing cell. Thus their association with the diplosome in the developing cilium is not surprising. Therefore, the basal body in the photoreceptor inner segment may function in the production of microtubules for the cilium, as does the centriole in spindle tubule formation. One centriole differentiates into a basal body while the other remains as the associated centriole. An electron-dense projection, basal foot, extends laterally from the mideentral region of the basal body during relocation distad. This basal foot process has also been referred to as a satellite arm (Sorokin, 1968) or fibrous tuft (Cohen, 1969). The number of foot processes per basal body is not known: however, our findings indicate that there are at least three in each photoreceptor cell in the retina. The banding pattern characteristic of the foot process was first described by deHarven and Bernhard (1956) in the respiratory tissue. Prior to its arrival at the plasmalemma, arm-like fibers form from one end of the basal body. These fibers appear to join to an electron-dense area. known as the basal plate [Fig. 9 (a)-(c)]. The vacuoles associated with the basal plate are similar to cytoplasmic vesicles described by Sorokin (1968). Sorokin suggested that these vesicles originate in the ctyoplasm as a result of an interaction between the basal body and the Golgi lamellae; the vacuoles are then thought to fuse with the plasmalemma. Upon arrival at the apex of the cell the basal plate seems to join with the plasma membrane, and the arm-like fibers appear to 'pull' the plasmalemma at the points of contact [Fig. 9 (d)-(f)]. The arm-like fibers that extend from the distal (apical) end of the basal body have not been described. Serial sectioning being done for a separate study may reveal the
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(a)
(b)
(
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(d)
(e)
(f)
(h)
(J)
(c)
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(g)
FIO. 9. Schematic representation of a proposed model of ciliogenesis in the retinal photoreceptor presented sequentially from left to right. (a) The diplosome resides in the apical cytoplasm ; one centriole (basal body) is attached to a basilar plate by arm-like fibers. The basilar plate borders an area of vaeuolated cytoplasm. (b) The basal body and basilar plate become closely associated with the plasmalemma. (c) The arm-like fibers 'tug' on the plasmalemma, initiating invagination. (d) The plasmalemma begins to invaginate, as the microtubules of the cilium begin to develop. (e)-(g) Invagination of the plasmalemma and lengthening of the cilium continues. (h) Oblique angle of developing cilium and the onset of disc formation. (i) Matured cilium with developing stack of disc membranes. precise m o r p h o l o g y of this structure. The arm-like fiber associated with the apex of the cell m a y create tension on the p l a s m a l e m m a resulting in an invagination of the cell m e m b r a n e [Fig. 9 (d)-(f)]. I t is i m p o r t a n t to remember t h a t each triplet of the basal b o d y has an arm-like fiber and thus invagination occurs in a ringlike fashion to produce a ' m o a t - l i k e ' appearance in appropriate cross-sections (Fig. 7). A similar form of invagination of the cell m e m b r a n e has been described in developing cilia of embryonic cartilage (Scherft and Daems, 1967), fetal rat lung (Sorokin, 1968), primordial upper respiratory tract (Friedmann and Bird, 1971), uterine endocervix (Philipp, 1977) and articular cartilage (Meier-Vismara, Walker and Vogel, 1979). At the same time t h a t the p l a s m a l e m m a invaginates at the apex of the cell body, doublet microtubules from each triplet elongate to form an evagination of the plasmalemma
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or ciliary bud. Further elongation of the ciliary bud results in the formation of the connecting cilium [Fig. 9 (g)-(i)]. The striated rootlet (ciliary rootlet) in ferret photoreceptors develops prior to or simultaneous with the invagination process and the development of the ciliary bud. In another study, Ash and Stephens (1975) reported that fully elongated cilia were present in molluscan gill filaments with only ' some indication' of the ciliary rootlets. This suggests that the ciliary rootlet grew late in the formation of the cilium in these specimens. The difference between our findings and theirs may be due to a species variation, variability in the plane of sectioning, or a high degree of variability from cell to cell. The striated rootlet in the mammalian photoreceptor cell may provide support for the basal body. Sebuwufu (1968) suggests that the main function of the rootlet is to anchor the cilium in the cell. The arm-like fibers described in this study may serve both supportive and anchoring functions by stabilizing the basal body. These arm-like fibers may be related to the anchoring system described by Sorokin (1962) in the fetal rat lung. Mattern, Daniel and Henkin (1970) reported nine radiating spokes in the ciliated cells of the tongue which may also be related to the arm-like fibers described in our study. Anderson (1972) observed fibers in the rhesus monkey oviduct that originate from the foot processes to reach the plasmalemma. An anchoring system binding the peripheral part of the basal body to the plasmalemma was described by Cordier (1975). Microtubules existing in a random fashion around the basal body may also contribute to supportive and anchoring functions. The developing cilium is covered by plasma membrane; therefore, as the cilium lengthens, there must be a concomitant increase in the plasma membrane. Our observations suggest that cytoplasmic vesicles fuse with the apical plasma membrane, thus resulting in an increase of the membrane. A similar phenomenon was suggested by Steinman (1968) and Komatsu and Fujita (1978a). This is supported by our observation that these smooth vesicles can be found fused with the plasma membrane in the region of the cilium; however, the direction of movement of these vesicles cannot be determined by electron micrographs alone. As the cilium lengthens, its distal end eventually projects past the apex of the cell [Fig. 9 (f) and (g)]. At first, only a few cilia extend into the potential space between the outer layer ofphotoreceptor cells and the pigment epithelium. The developing cilia abut the apposing pigmented epithelium. As described here and in our previous studies of the mammalian retina (Weidman and Kuwabara, 1968; Greiner and Weidman, 1978, 1980), the developing cilium initially grows at an angle; it later straightens to become oriented parallel to the long axis of the cell. We believe that the opposing force presented by the pigmented epithelium causes the cilium to develop initially at an angle to the longitudinal axis of the photoreceptor cell. However, as cilium development proceeds and becomes more or less synchronous, the force exerted by the growth and straightening of a large number of cilia increases the distance between the pigment epithelium and the photoreceptor cell bodies, allowing initial straight growth of later-developing cilia. The morphologie evidence presented herein supports the hypothesis that ciliogenesis is complete when disc membranes form at the distal end of the cilium, resulting in the development of the outer segment.
Comparative ciliogenesis Unlike most ciliated cells, which have multiple motile cilia, the photoreceptor cell of the retina has a single nonmotile cilium. The cell with multiple cilia has a solitary
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centriole (basal body) associated with each cilium. Single (primary) cilia have a diplosome consisting of one basal body and of one associated centriole. In cross-section the single cilium lacks the central pair of ciliary fibers (a 9 + 0 microtubular pattern) common to a motile cilium. Single cilia occur in sensory cells such as retinal photoreceptors and vestibular hair cells as well as in a number of undifferentiated cells which are nonciliated when mature. The transitory existence of the latter has been described by Sorokiu (1968). Cells with a single cilium have been reported in mammalian tissues such as connective tissue (Meier-Vismara et al., 1979), glandular tissue (Kamatsu and Fujita, 1978b), muscle (Przybylski, 1971), kidney (Webber and Lee, !975), and nervous tissue (del Carro and Snider, 1969}. Frequently these cilia are found in the cytoplasm or in vacuoles or invagiuatious of the plasmalemma, the nuclear membrane and elsewhere (Friedmann and Bird, 1971). Scherft and DaemS (1967) suggest ' t h a t the potency to form single cilia is present in almost every kind of tissue', but the significance of most of these cilia is still obscure. For example, in describing the single cilium of the articular cartilage of the cat, Meier-Vismara et al. (1979) observed the majority of the single cilia undergoing involution. Although in most cells solitary cilia are transitory and tend to decrease in number with maturation, these structures sometimes persist in the fully differentiated cells, e.g. secretory cells (Komatsu and Fuj!ta, 1978b) and sensory cells such as the retinal photoreceptor, in the retinal photoreceptor the early embryonic cilium does not involute but serves as the connecting piece between the inner and outer segments. Ciliary rootlets are present in both singly and multiply ciliated cells. However, the shape of these rootlets varies among cell types. In contrast to the multiciliated cell, the ciliary rootlets of the photoreceptor cells and other singly ciliated cells do not diverge as widely as they extend basally in the cell cytoplasm. Solitary cilia in many epithelia undergo a similar developmental process as in the retinal photoreceptor cilium. In fully developed retinal photoreceptors, the connecting cilia are uniform in length. Sorokin's (1968) observation that primary cilia of the epithelial border of the lung are of variable length may be an artifact of sectioning, since these cilia are randomly oriented. Thus it appears that the 'primary cilium' of retinal photoreceptors does fall into the rudimentary category developmentally but instead of involuting the primary cilium remains throughout life and serves to connect the inner and outer segments of the photor~eeptor. This has great significance for what is known concerning disc membrane production and synthesis, and transport of biologically active components of the photoreceptor membrane. ACKNOWLEDGMENTS This study was supported by Institutional National Research Service Award EY-07018 awarded to the Eye Research Institute and by core grant EY-01792 to the Department of Ophthalmology, University of Illinois by the National Eye Institute, National Institutes of Health. The authors thank Ms Ruth Zelkha for photography and Ms Bettye Harris and Mrs Marjorie Pole for the typing of the manuscript. REFERENCES Anderson, R. G. W. (1972). The three dimensional structure of the basal body from the rhesus monkey oviduct. J. Cell Biol. 54, 246-65. Ash, B.M. and Stephens, R.E. (1975). Ciliogenesis during the sequential formation of Molluscan gill filaments. Dev. Biol. 43, 340-7.
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del Cerro, M. P. and Snider, R. S. (1969). The Purkinje cell cilium. Anat. Rec. 165, 127-40. Cohen, A. I. (1969). Rods and cones and the problem of visual excitation. In The Retina. Morphology, Function and Clinical Characteristics (Eds Straatsma, B. R., Hall, M. 0., Allen, R. A. and Creseitelli, F.). Pp. 31-72. University of California Press, Berkeley and Los Angeles. Cordier, A. C. (1975). Ciliogenesis and ciliary anomalies in thymic cysts o f ' n u d e ' mice. Cell Tissue Res. 148, 397-406. DeRobertis, E. (1956). Morphogenesis of the retinal rods. J. Biophys. Biochem. Cytol. 2 (Supp.), 209-18. Friedmann, I. and Bird, E. S. (1971). Ciliary structure, ciliogenesis, microvilli. Electron" microscopy of the mucosa of the upper respiratory tract. Laryngoscope 81, 1852-68. Greiner, J. V. and Weidman, T. A. (1978). Development of the hamster retina. A morphologic study. Am. J. Vet. Res. 39, 665-70. Greiner, J. V. and Weidmann, T. A. (1980). Histogenesis of the eat retina. Exp. Eye Res. 30, 439-53. Greiner, J. V. and Weidmann, T. A. (1981). Histogenesis of the ferret retina. Exp. Eye Res. 33, 315-32. deHarven, E. and Bernhard, W. (1956). Etude au microscope 61ectronique de 1'ultrastructure du centriole chez les vert6br6s. Z. Zellforsch. Mikrosk. Anat. 45,378-98. Komatsu, M. and Fujita, H. (1978a). Some observations on eiliogenesis in the oviduct epithelium of the mouse. Arch. Histol. Jpn 41,229-37. Komatsu, M. and Fujita, H. (1978b). Electron-microscopic studies on the development and aging of the oviduct epithelium of mice. Anat. Embryol. 152, 243-59. Mattern, C. F. T., Daniel, W. A. and Henkin, R. I. (1970). The ultrastructure of the htiman circumvallate papilla. I. Cilia of the papillary crypt. Anat. Rec. I67, 175-82. Meier-Vismara, E., Walker, N. and Vogel, A. (1979). Single cilia in the articular cartilage of the cat. Exp. Cell Biol. 47, 161-71. Philipp, E. (1977). Elektronenmikroskopische Untersuchiangen fiber die Ciliogenese in Flimmerzellen der menschliehen Endocervix uteri. Med. Welt 28, 563-68. Przybylski, R. J. (1971). Occurence of centrioles during skeletal and cardiac myogenesis. J. Cell Biol. 48, 214-21. Scherft, J. P. and Daems, W. T. (1967). Single cilia in chondrocytes. J. Ultrastruct. Res. 19, 546-55. Sebuwufu, P. H. (1968). Ultrastructure of human fetal thymic cilia. J. Ultrastruct. Res. 24, 171-80.
Sorokin, S. (1962). Centrioles and the formation of rudimentary cilia by fibroblasts and smooth muscle cells. J. Cell Biol. 15,363-77. Sorokin, S. P. (1968). Reconstructions of centriole formation and ciliogenesis in mammalian lungs. J. Cell Sci. 3, 207-30. Staprans, I. and Dirksen, E. R. (1974). Microtubule protein during eiliogenesis in the mouse oviduct. J. Cell Biol. 52, 164-74. Steinman, R. M. (1968). An electron microscopic study of ciliogenesis in developing epidermis and trachea in the embryo of Xenopus laevis. Am. J. Anat. 122, 19-56. Tokuyasu, K. and Yamada, E. (1959). The fine structure of the retina studied with the electron microscope. IV. Morphogenesis of outer segments of retinal rods. J. Biophys. Biochem. Cytol. 5, 225-30. Webber, W. A. and Lee, J. (1975). Fine structure of mammalian renal cilia. Anat. Rec. 182, 339-44. Weidman, T. A. and Kuwabara, T. (1968). Postnatal development of the rat retina. An electron microscopic study. Arch. Ophthalmol. 79, 470-84.