Cell Tissue Res. 199, 319-338 (1979)
Cell and Tissue Research 9 by Springer-Verlag 1979
The Fine Structure of the Hypostome and Mouth of Hydra II. Transmission Electron Microscopy Richard L. W o o d Department of Anatomy University of Southern California School of Medicine, Los Angeles, USA
Summary. The normal morphology of the hypostome and mouth of hydra were examined by transmission electron microscopy with conventional thin sections and freeze-fracture replicas. Myonemes of the hypostome are small in diameter, have gap and intermediate-type cell junctions within each epithelial layer and are associated with the opposite epithelial layer by transmesogleal processes and gap junctions. Nematocysts and sensory cells are aggregated in the circumoral region. The fine structure of adherent flagella arising from gastrodermal gland cells, and the transition region at the mouth between epidermis and gastrodermis are described in detail for the first time. The possible functional significance of the findings is discussed. Key words: H y d r a - H y p o s t o m e - Myonemes - Flagella - Freeze-fracture
As indicated in the companion paper (Wood, 1979) the hypostome of hydra has a central role in m a n y physiological activities. In the adult this region is involved with highly complex movements during capture and ingestion of prey, and during development and regeneration it is involved with the establishment of the polarity of the differentiating polyp. In order to understand better the mechanism of mouth opening, the process of engulfment and, eventually, the sequence of morphological changes in hypostome differentiation, it seemed essential to examine the fine structure of the entire hypostome of normal animals in greater detail. This paper presents the results of studies of thin sections and freeze-fracture replicas from the hypostome of normal adult hydra. Send offprint requests to: Richard L. Wood, Ph.D. Department of Anatomy University of Southern
California School of Medicine, 2025 Zonal Avenue Los Angeles, California 90033, USA Acknowledgements. The technical assistanceof Ms. AileenKuda and Mr. Watchara Pimprapaiporn and suggestions for improvingthe manuscript by Dr. Douglas Kellyare gratefullyacknowledged.Supported by grants PCM 77-14635 from the National ScienceFoundation and by funds from the LivingStructure Fund of the Department of Anatomy
0302-766X/79/0199/0319/$04.00
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Materials and Methods Cultures of Hydra littoralis and Hydra attenuata were maintained in the laboratory by the methods of Loomis and Lenhoff (1956) or Lenhoff and Brown (1970). Animals were anesthetized and fixed in most cases by the methods described in the previous paper (Wood, 1979) for routine thin sectioning, and were fixed in 0.75 ~ glutaraldehyde plus 0.75 ~ formaldehyde in 0.05 M cacodylate buffer, pH 7.4, for 1530 rain for freeze-fracture. In some instances sodium phosphate was used for buffering and either 2 ~o tannic acid or a few drops of 30 ~oH202 were added to the primary aldehyde fixatives (Peracchia and Mittler, 1972). Glutathione-treated animals were fixed without anesthetization. Specimens were embedded either in Araldite 502 (Wood, 1977) or in Spurr's low viscosity resin (Spurr, 1969). Blocks were mounted for either cross or longitudinal sectioning using a Porter-Blum IIB ultramicrotome. One-lma sections were stained with methylene-blue-Azure II (Richardson et al., 1960), mounted in immersion oil and photographed with a Leitz Ortholux microscope. Thin sections were collected on carbon-coated grids and stained with uranyl acetate/lead citrate to enhance contrast. For freeze-fracture, hypostomes of aldehyde-fixed animals were removed from the body stalk, trimmed free of tentacles under a dissecting microscope and either sliced in half longitudinally with a razor blade by hand or embedded in 3 ~ agar and chopped at 100 txm with a Sorvall TC-2 tissue chopper. Tissue slices were then glycerinated for 20-30rain each in 10~, 20~ and 30~o glycerol in 0.05 M cacodylate buffer, placed on gold alloy specimen discs and frozen in liquid Freon 22 held near its freezing point with liquid nitrogen. Fracturing and replication were accomplished in a Balzers 301 apparatus equipped with an electron gun for platinum-carbon shadowing and a film thickness monitor. Replicas were cleaned in methyl alcohol, household bleach and sulfuric acid, rinsed in distilled water and picked up on formvar-coated, 75-mesh copper grids. Thin sections and replicas were examined and photographed with a JEOL 100-C electron microscope.
Observations The h y p o s t o m e , like o t h e r b o d y regions, consists o f two epithelial layers s e p a r a t e d b y a thin m e s o g l e a ; a n i n t e r r u p t i o n in this p a t t e r n occurs at the m o u t h . I n the following account, the two epithelial layers a n d the m o u t h region are described separately.
Epidermis I n relaxed a n i m a l s the e p i d e r m i s is thin over m o s t o f the h y p o s t o m e (Fig. 1). Relatively large, e p i t h e l i o m u s c u l a r cells f o r m a n e a r l y c o n t i n u o u s layer in which n e m a t o c y t e s , n e m a t o b l a s t s , interstitial cells a n d nerve cells are intercalated. The apices o f e p i t h e l i o m u s c u l a r cells are a t t a c h e d to each o t h e r b y septate j u n c t i o n s a n d there are n u m e r o u s gap j u n c t i o n s a l o n g their lateral surfaces. W i t h the exception o f m a t u r e n e m a t o c y t e s a n d sensory cells, which are a t t a c h e d apically to epit h e l i o m u s c u l a r cells by septate junctions, n o n e o f the o t h e r cell types possess well d e v e l o p e d cell junctions. The e p i t h e l i o m u s c u l a r cells c o n t a i n v a r i o u s sized vacuoles a n d there is a layer o f irregularly s h a p e d granules u n d e r l y i n g the external surface. T h e granules m a y a p p e a r either dense o r lucent d e p e n d i n g on the m e t h o d o f fixation (Figs. 2 a n d 3). The free surface o f e p i d e r m a l cells is covered b y a cuticular layer, p r e s u m a b l y derived f r o m the c o n t e n t s o f the subsurface granules. In freeze-fracture p r e p a r a t i o n s the m e m b r a n e at the free surface o f e p i d e r m a l cells c o n t a i n s closely p a c k e d i n t r a m e m b r a n o u s particle a r r a y s o n the P fracture face a n d c o m p l e m e n t a r y a r r a y s o f pits o n the E fracture face (Figs. 4 a n d 5).
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Fig. 1.a 1 ~m parasagittal section of hypostome. The arrow indicates a nematocyst, x 380 The basal region of epitheliomuscular cells is drawn out into multiple myoid processes (myonemes) which run longitudinally (radially if viewed from above the mouth). This corresponds to the arrangement of epidermal myonemes in other body regions. Measurements were made of cross sectional diameters of 100 consecutive myonemes in a randomly selected section through each of four different body regions of a well expanded, large animal. As can be seen from the summary of these data in Table 1, the average size of the myonemes of the distal hypostome is smaller than those of the tentacles and proximal hypostome. The differences are highly significant statistically (p < 0.0005, Student's t-test). When apical hypostome and gastric region myonemes were compared there was no significant difference. Myonemes are attached to each other end-to-end, and to some extent on lateral surfaces, by intermediate-type intercellular junctions (Figs. 6 and 7). Interdigitated myonemes are also joined laterally by gap junctions (Figs. 6 and 7, arrows). In addition, small cytoplasmic processes extending across the mesoglea make gap junctional contact between epidermal and gastrodermal cells (Fig. 8, arrow). Counts of the number of transmesogleal contacts per linear extent of mesoglea 1
Unlessotherwiseindicated, all figures are from Hydra attenuata
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Fig. 2. Epidermis of hypostome of Hydra littoralis after combined aldehyde-OsO 4 fixation. C eutical layer; G subsurface granules; N nerve cell. x 7300 Fig. 3. Epidermis of hypostome of Hydra littoralis after sequential aldehyde-OsO 4 fixation. Labels as in Fig. 2. • 5500 Fig. 4. Intramembranous particle arrays on P face of epidermal cell. x 125,500 Fig. 5. Complementary pit arrays on E face of epidermal cell. x 125,500
Table 1. Comparison of myoneme sizes in different body regions
Region
Number of measurementsa
Average area in Ixm2
S.E.
Tentacles Distal hypostome Proximal hypostome Gastric region (column wall)
100 100 100 100
1.54 0.65 0.92 0.66
0.089 0.043 0.054 0.036
a For these purposes the myonemes were measured in cross sections from micrographs taken at 2,700X and enlarged to 6,750X. The assumption was made that the myonemes were square or rectangular and area was calculated as equal to average length X average width. All myonemes in a field were counted and fields were selected randomly
Fig. 6. Epidermal myonemes show intermediate junction (right) and gap junction (arrow)joining lateral surfaces. N neurite. • 47,500 Fig. 7. End-to-end junction of epidermal myonemes showing intermediate junction. Arrows indicate gap junctions, x 46,500
Fig. 8. Transmesogleal gap junction in Hydra littoralis (arrow). GS gastrodermal cell; M mesoglea; N nerve cell in epidermis, x 15,500 Figs. 9 and 10. P- and E-face images of mesogleal surface membranes of epidermal cells showing focal specialization. • 62,500 Fig. 11. 1 ~tm section showing aggregation of sensory cells (arrows) and ganglion cells near mouth
(GAN). NEM nematoblasts, x 320
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2. Number of transmesoglealcontactsa in different body regions
Region
Distance along mesoglea Numberof Number in ~tm contacts ~rn
Tentacles Distal hypostome Proximal hypostome Gastric region
200 220 200 194
32 113 50 48
0.16 0.51 0.25 0.25
a For these data, all obvious transmesoglealprocesseswere counted even where actual contact was not observed within the section. Sections of approximately0.1 ~tm thickness were used throughout
show that such contacts are most numerous in the distal hypostome, (Table 2). The difference in number between distal and proximal hypostome is a factor of 2 and between distal hypostome and tentacles is a factor of 4. Not surprisingly, the number of contacts was the same in the gastric region as in the proximal hypostome. Epidermal myonemes in all body regions appear to have a distinctive feature when viewed by freeze-fracture. Small clusters of intramembranous particles appear on elevations on the P face of the cell membrane where it is apposed to the mesoglea (Fig. 9). A complementary depression occurs on the E face of the same membrane (Fig. 10); some such depressions display arrays of complementary pits. The significance of this intramembranous specialization is not entirely clear but it may reflect regions of firm attachment to the mesoglea. Nematocysts and nematoblasts are present in the epidermis of the hypostome, particularly in the circumoral region (Figs. 1 and 11). They occur within nematocytes that are embedded in epitheliomuscular cells. Nematocytes in the hypostome are not arranged in batteries as is characteristic for the tentacles. The freeze-fracture morphology of hypostomal nematocysts does not differ remarkably from that of tentacle nematocysts; the latter will be the subject of a separate communication. The hypostomal epidermis contains many small basally located cells. Some of these are clearly nematoblasts and interstitial cells, but most appear to be nerve cells (Figs. 2, 3, 11, 12 and 13). Neurites are represented by numerous small cell processes containing either groups ofmicrotubules or clusters of 80-250 nm vesicles, many of which have dense cores (Fig. 13). Since nerve cells of hydra have been studied extensively by other investigators (Burnett et al., 1964; Lentz, 1965; Davis et al., 1968; Bode et al., 1973; Westfall and Townsend, 1976; Epp and Tardent, 1978; Westfall and Kinnamon, 1978) the details of their fine structural morphology will not be reiterated here. It should be noted, however, that the present observations do demonstrate an aggregation of sensory cells in the circumoral region (Fig. 11). The morphology of these sensory cells appears similar to those of the tentacles as described recently by Westfall and Kinnamon (1978). An apical club-shaped cilium is present but it rests in a surface depression and does not extend above the general cell surfaces (Fig. 13).
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Fig. 12. Interstitial cell in epidermis of Hydra titwralis. E external surface; M mesoglea. • 5500 Fig. 13. Sensory cell reaching epidermal surface near mouth. SC sensory cilium; M mesoglea; N neurite with dense cored vesicles, x 5900
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Fig. 14. Longitudinal section of hypostome, mouth at top of field. G1 and G2, gland cells with two granule types; D digestive cells with microvilli borders; V phagocytic vacuoles. • 5400
Gastrodermis Gastrodermal cells of the hypostome are predominantly glandular. Most of the cells contain large secretory granules of irregular density but a few contain smaller dense granules. It is not certain whether these represent different cell types or merely different secretory phases of the same cell. Some non-glandular digestive cells are also present (Fig. 14,D). Both glandular and digestive cells possess flagella. It is clear from both thin sections and freeze-fracture replicas that flagella of the glandular cell are directed
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aborally and lie in close proximity to the luminal surfaces of the gastrodermal cells(Figs. 14, 15 and 16). A few flagella in the hypostome extend freely into the lumen; it is presumed that these arise from the digestive cells but their low frequency has made it difficult to determine their origin conclusively. Except for flagella, the glandular cell surfaces are relatively smooth, whereas digestive cell surfaces are covered with lamellar folds and elongate microvilli. The digestive cells also contain phagocytic vacuoles of various sizes. The flagella of glandular cells arise in pairs from typical basal bodies but more than one pair may arise from a single cell. The proximal region of the flagellum is constricted slightly where the basal body terminates and the axoneme commences (Fig. 16). The narrowest part of the constriction measures about 0.2 gm in diameter in thin sections. Beyond the constriction the flagellum expands slightly and the surface membrane assumes a scalloped or fluted appearance. More distally the flagellar cytoplasm expands towards the underlying cell and the axoneme remains eccentrically located along the free edge (Fig. 16). Cross sections show that fluting begins at the junction between basal body and flagellar shaft and continues onto the shaft proper (Fig. 17). There are nine flutes, each associated with a peripheral doublet by a density having a champagne glass configuration (Figs. 17 and 18) as has been described for lamellibranch cilia (Gilula and Satir, 1972). Cross sections also reveal that the more distal portion of the flagellum has wing-like lateral extensions that may be up to 0.8 l~m in total width (Fig. 19). The lateral extensions are smaller in the distal half of the flagellar shafts but the basic appearance is similar except at the extreme distal tip. Both transverse and longitudinal sections show that the expanded flagellar cytoplasm contains a condensation of filamentous material that appears to maintain an association with the peripheral doublets of the axoneme by delicate strands of dense material (Figs. 16, 19 and 20). The expanded surface of the flagellum lies in a groove along the surface of the underlying cell but the two plasma membranes remain separated by a distance of 25-30nm. The cytoplasm of the underlying cell, as well as the 25-30 nm intervening space, may also contain electron-dense material (Figs. 16 and 20). This special relationship to underlying cells occurs over the entire flagellar length, with the exception that the close apposition is lost where the flagella pass from one cell surface to the next, and at the distalmost tip. In freeze-fracture replicas the most proximal portion of the flagellum displays a 2-3 stranded neclace of P-face intramembranous particles (Figs. 21 and 22). The Fig. 15. E-faceviewof lumenalsurfaceof gastrodermalcell showingflagellarpairs runningin grooves.
x 23,200 Fig. 16. Proximal portion of adherent flagellum.B basal body; A axoneme; F filamentousmaterial connectingaxonemewithflagellarsurfaceapposedto underlyingcell.X, Y, Z, sectionlevelsfor Figures 17, 18, and 19. x 32,250 Fig. 17. Section through basal plate (X Fig. 16) of adherent flagellum. • 152,000 Fig. 18. Sectionthrough fluted region (Y Fig. 16) of adherent flagellum, x 152,000 Fig. 19. Section through shafts (Z Fig. 16) of adherent flagella.Note density of flagellar-cellsurface interspace, I. x 103,000
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Fig. 20. Longitudinal section of adherent flagellum (top) apposed to underlying cell (Hydra littoralis). Note filamentous condensation between axoneme and flagellar surface (arrows). x 98,300 Fig. 21. Proximal portion of flagellum showing necklace and fluted region, P face image, x 43,400 Fig. 22. Proximal flagellum showing P face particle clusters on membrane flutings (arrows). x 80,000 Fig. 23. Proximal flagellum E-face image of fluted region. • 52,500
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adjacent basal body region may also have an aggregation of 12.5-15 nm P-face particles. Distal to the flagellar necklace, the flagellar shaft narrows to about 0.2 I~m in diameter and then expands into a fluted region about 0.3-0.4 ~m in diameter and up to 0.6 lam long. The P-face ridges on the fluted portion have clusters of 12.5 nm particles and the interposed grooves are nearly particle-free (Figs. 21 and 22). The E face image of the fluted portion shows a few 10-12.5 nm particles in the grooves (complementary to the P face ridges) and particle-free ridges (Fig. 23). Beyond the fluted portion, the flagellar shaft is somewhat irregular in diameter; the portion housing the axoneme averages about 0.3 ~tm in width and the expanded wings range from 0.44).8 ~tm in total width. The indentations of the underlying cells, along which the flagella run, match the width of the expanded wings (Fig. 15). The shaft of the flagellum has typical 12.5nm intramembranous particles, predominantly on the P face, and some 8-10nm low profile particles (Fig. 21). Particle distribution appears to be random over most of the length of the free border of the shaft, although increased numbers of 12.5nm particles appear on some flagella proximally. The latter may be indicative of forming flagella. By contrast, the expanded portion facing the underlying cells has few 12.5 nm particles but has 8-10nm particles (Fig. 24, arrow). This pattern of particle distribution occurs on both P and E faces of the expanded wings and on P and E faces of the matching groove in the underlying cell. Absorptive cells have numerous microvilli on their lumenal surfaces and also may have several closely apposed flagella associated with these surfaces. All gastrodermal cells reaching the lumen are attached to each other by septate junctions apically and there are numerous gap junctions along lateral cell surfaces. Basally, some gastrodermal cells possess myonemes that course circumferentially. As noted above, gastrodermal myonemes extend small cytoplasmic processes into and through the mesoglea to make gap junctional contact with the myonemes of the epidermal layer. Gastrodermal myonemes do not possess the small clusters of Pface intramembranous particle arrays on their mesogleal surfaces that are characteristic of epidermal myonemes.
Mouth
The mouth region is characterized morphologically as a zone of transition between epidermis and gastrodermis. Although it can be identified in non-feeding animals, the details of the arrangement of cells in the transition zone is most readily studied in animals with open mouths. As noted by scanning electron microscopy in the preceding paper (Wood, 1979), the boundary between the two epithelial layers is marked by a depression or groove underlain by transitional cells (Fig. 25). Most of the transitional cells do not possess typical features of gastrodermal cells, such as secretion granules or flagella, nor do they contain subsurface granules or have the external cuticular layer characteristic of epidermal cells. The transitional cells are attached to each other and to adjacent epidermal and gastrodermal cells by both septate and gap junctions. Smaller, basally located nerve cells lacking obvious junctional specializations are also present near the mouth, as are neurites containing dense-core vesicles
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Fig. 24. E face of gastrodermal cell lumenal surface, L and P-face of flagellum (arrow). • 54,500 Fig. 25. Transitional area at edge of open mouth, Hydra littoralis. The flagella in the transitional groove (left) are gastrodermal in origin. C cuticular layer; E epidermal cell; G gastrodermal gland cell; T transitional cells; M mesoglea; N neurite; V phagocytic vacuoles, x 8700 Fig. 26. P face image of transitional cell external surface. Intramembrane particle aggregates (arrows) are small in size and sparse in number, x 55,600
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(Fig. 25). As noted previously there is also an aggregation of spindle-shaped sensory cells in the circumoral region. Sensory cells are attached apically to adjacent epitheliomuscular cells by septate junctions. The mesoglea is attenuated and terminates at a variable distance from the mouth edge, allowing extensive direct contact between epidermal, gastrodermal and transitional cells (Fig. 25). There are broad regions of the latter cellular contacts that lack obvious junctional specializations. Myonemes of both epidermal and gastrodermal cells become small and irregularly disposed in the area of mesoglea termination. The transitional cells do not possess recognizable myonemes. Freeze-fracture replicas show that the transitional region also exhibits a gradation in membrane morphology. Surface membranes of epidermal cells near the mouth show a reduction in the number and size of P-face particle arrays (Fig. 26) (Compare with Fig. 4) and there is a total absence of the particle arrays on some of the transitional cell surface membranes. The particle arrays have never been observed on gastrodermal cells.
Discussion
This study has provided a considerable amount of new information that adds to our understanding of the organization of the hypostome as it may relate to general functions. It also provides a basis for interpreting the results of studies now in progress on the formation of the hypostome during regeneration. The most pertinent findings will be discussed under the headings: (1) Hypostomal Movement, (2) Transitional Zone, and (3) Adherent Flagella.
Hypostomal Movement In observing the feeding activity of hydra, the complexity of coordinated movements of the hypostome and tentacles is striking. It seems obvious that there must be extensive interaction between circularly and longitudinally oriented myonemes of the two epithelial layers and mechanisms for fine control of these interactions must exist. Three morphological features of the hypostome are candidates for involvement in such fine control: (1) The hypostome has the greatest number of nerve cells of any body region. (2) The number of contacts between epidermal and gastrodermal myonemes and, concomitantly, the number of gap junctions involving cell processes from the two epithelial layers, is larger in the hypostome than in any other body region. (3) The average size ofmyonemes (crosssectional diameter) is small in the hypostome and decreases apically. Bode and coworkers (1973) have determined that over 20 % of the cells of the hypostome of Hydra attenuata are neural cells. The only other body region that has an equivalent relative number of neural cells is the basal disc. Since there is a well documented developmental gradient centered in the head region of hydra (Shostak, 1974), it has been advocated that neural cells are the source of diffusible substances involved with this gradient (Lentz, 1965). Recent evidence that nerve-free hydra can regenerate normally indicates that the developmental gradient cannot be
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maintained by neural cells alone (Marcum et al., 1977). Although the possibility that neural cells might serve as a source of morphogens has understandably excited attention, the high numbers of these cells in the hypostome suggests that they probably play an important role in more routine physiological activities as well. In fact, physiological studies have provided evidence that the major function of hydra neural cells may relate to the initiation of spontaneous contractions of the musculature (Passano and McCullough, 1962). An additional function may be involvement in fine control of localized movements. The fact that nerve-free hydra do not exhibit normal hypostomal activity but must be fed by placing food material into the enteron directly (Campbell, 1976) is consistent with such a function. The distribution of neural cells on the hypostome has not been completely elucidated. One point in question is whether or not there is a concentration of specialized sensory components in the circumoral region. It was noted in the previous paper (Wood, 1979) that cnidocils and other types of sensory hairs could not be distinguished morphologically on the hypostome in our scanning electron micrographs. However, transmission electron microscopy has confirmed that ciliated sensory cells similar to those recently described for the tentacles (Westfall and Kinnamon, 1978) do occur on the hypostome and they appear to be more numerous around the mouth. This is not surprising in view of the normal sequence of events during feeding where the mouth opens as prey organisms are drawn to the hypostomal surface by tentacular contraction. The fact that mouth opening can be induced chemically suggests that activation of the circumoral sensory cells is at least partially a chemically mediated phenomenon. A second feature of the hypostomal region that seems likely to be associated with precise localized movements is the extensive gap-junctional contact within each epithelial layer and across the mesoglea (Wood, 1977). Although there is no direct evidence that gap junctions of hydra are involved with intercellular communication, the electrical properties of the body wall in normal (Josephson and Macklin, 1967 and 1969) and nerve-free animals (Campbell et al., 1976) coupled with the mounting evidence that gap junctions serve this role in other systems 9(Gilula, 1977), support this assumption. Thus, increased numbers of gap junctions would be generally regarded as evidence indicating an increase in intercellular communication. Of particular significance in this regard is the large number of gapjunctional contacts in the hypostome between myonemes situated on opposite sides of the supporting mesoglea. This would be predicted if gap junctions were indeed involved in fine coordination of contractile activity of the two muscular layers in this region. There is scant morphological evidence that neurites traverse the mesoglea so it seems likely that such coordination must be accomplished, at least in part, by the gap junctional contacts. It is of interest, however, that the interaction between circularly and longitudinally oriented myonemes cannot simply be the induction of a comparable simultaneous activity. Coordination must involve opposing and independent activity of the two layers. That this is probably so is supported by the observation that the two layers of myonemes respond differently to urethane anesthesia (Macklin, 1976). Thus, the mechanism of transmesogleal contractile coordination is not immediately obvious, but a role for the numerous gap junctions seems likely. The third feature of hypostomal organization that may be important in fine control of movement is the size of individual epidermal myonemes. Smaller
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myonemes would be expected to result in less powerful contraction, but would also facilitate more delicate local movements if one assumes that the myonemes are capable of independent activity and that the larger numbers of neurons in the hypostome reflect increased innervation of myonemes. The present observations are consistent with this concept. Other factors such as relative lengths of each myoneme, or number of myonemes per epithelial cell, also would have an effect on fine control of movement, but quantitative evidence on these features is difficult to obtain because of sampling problems.
Transition Zone The cells of the transition zone at the mouth edge have morphological features different from typical gastrodermal and epidermal cells. Aside from representing a transition in morphology from gastrodermis to epidermis, it seems likely that the region has other functional significance. One possibility is that some of the transitional cells represent cells being lost in normal cell turnover by degeneration or sloughing. Kinetic studies of cell turnover in hydra (Campbell, 1967) have shown that cells are lost at the extremities, including the apex of the hypostome. The present observations show that phagocytic vacuoles containing cellular debris are present in some of the transitional cells. The present preparations did not show images that could be interpreted as sloughing cells, but such views could have been missed because of sampling difficulties inherent with thin sectioning techniques. A second possibility is that the transitional zone has a special sensory function. The cells that reach the surface do not appear to be degenerating but do differ in morphology from other epithelial cells in the vicinity. Although they do not appear to possess ciliary specializations, they could still be sensory in nature. Typical sensory and ganglion cells are present close by with which the transitional cells could be interacting. There is no evidence at this time that the transitional area represents a proliferative zone for nematocytes or any other cell type and the transitional cells have not been observed to be undergoing mitosis. However, this does not exclude the possibility that they may be differentiating sensory cells, a possibility that deserves further attention.
Adherent Flagella A major contribution of the present study is the elucidation of the fine structural morphology of the adherent flagellum. The close relationship of these flagella to underlying cells noted previously by scanning electron microscopy (Westfall and Townsend, 1977; Wood, 1979) is confirmed in the present study and further details are reported. The evidence that these flagella are truly adherent, and thus relatively immotile, is indirect, but taken in total perspective the evidence is fairly compelling. (1) These flagella appear to arise from only one cell type and to be directed invariably toward the base of the hypostome. (2) The expanded wings of cytoplasm characteristic of these flagella lie in grooves along the apices of gastrodermal cells, and the close apposition of the two plasma membranes is reminiscent of the relationship of flagella to cell surfaces in trypanosomes (Vickerman, 1969; Smith et al., 1974) for
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which flagellar adhesion is well documented. In the latter case, there are regularly disposed, small junctional complexes along the flagellar groove that are not present in hydra, but it has been suggested that these complexes may be involved with anchoring of intercellular components to the cell surface rather than with actual flagellar attachment (Smith et al., 1974). (3) Adherent flagella of hydra have filamentous condensations in the expanded portion facing the underlying cells, as does the subjacent cytoplasm of the latter cells. The intervening intercellular space is of constant width and also contains electron dense material. (4) Intramembranous particle distribution differs from other regions of the same membranes for both the flagellar wings and the underlying grooves. The reduction in numbers of regular sized particles (12.5 nm in this case) is reminiscent of the intramembranous appearance of intermediate-type junctions in higher organisms (McNutt, 1977). All of these features indicate a special functional relationship between these flagella and the underlying cells and are strongly suggestive of adhesion with at least partial immobilization of flagellar movement. The morphology of the proximal part of the adherent flagella is also noteworthy. As with most other flagella and cilia so far examined, a ciliary necklace occurs around an initial conical segment. This is followed by a region of prominent fluting of the membrane which extends for a variable distance onto the flagellar shaft. The flutes are precisely arranged with respect to the peripheral doublets of the axoneme and, apparently are anchored to the latter by dense strands that end within the membrane in small aggregates of 12.5 nm intramembranous particles. This configuration seems most similar to what has been described for gill cilia of clams (Gilula and Satir, 1972) but also bears some resemblance to the granule plaques of the cilia of Paramecium (Plattner, 1975). For these cilia it is suggested that intramembranous particle aggregates associated with the basal body region are involved in binding and exchange of calcium ions (Plattner, 1975), but this possibility has not been explored for hydra. The biological function of adherent flagella in the hypostome of hydra can only be speculated upon at this time. The axoneme appears normal in organization insofar as it has been examined to date. It is possible that these flagella simply serve to protect the lumenal surfaces ofgastrodermal cells by helping to hold a coating of mucus, as suggested by Westfall and Townsend (1977), but the close relationship to underlying cells demonstrated here suggests a more complex function. In trypanosomes, adherent flagella initiate undulatory movements of whole cells and it is possible that this kind of movement of lumenal surfaces of gastrodermal cells could occur in the hypostome of hydra. This possibility deserves consideration since in normal feeding behavior swallowing appears to involve an active creeping of the open hypostome around the prey organism rather than the mouth merely serving as a receptacle for material being pushed downward by the tentacles. Adherent flagella could play an important role in conjunction with myoneme contractions that implement creeping motion. This hypothesis is readily testable and experiments are being designed to study these flagella more extensively. References
Bode, H., Berking,S., David, C.N., Gierer,A., Schaller, H., Trenkner, E.: Quantitativeanalysisof cell types during growth and morphogenesis in Hydra. Wilhelm Roux Arch. Entwicklungsmech. Organismen 171, 269-285 (1973)
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Burnett, A.L., Diehl, N.A., Diehl, F.: The nervous system of hydra. II. Control of growth and regeneration by neurosecretory cells. J. Exp. Zool. 157, 227-236 (1964) Campbell, R.D.: Tissue dynamics of steady state growth in Hydra littoralis. II. Patterns of tissue movement. J. Morph. 121, 19-28 (1967) Campbell, R.D.: Elimination of Hydra interstitial and nerve cells by means of colchicine. J. Cell. Cci. 21, 1-13 (1976) Campbell, R. D., Josephson, R,K., Schwab, W.E., Rushforth, N.B.: Excitability of nerve-free Hydra. Nature 2,62, 338-340 (1976) Davis, L., Burnett, A., Haynes, J.: Histological and ultrastructural study of the muscular and nervous systems in Hydra. II. Nervous system. J. Exp. Zool. 167, 295-332 (1968) Epp, L., Tardent, P.: The distribution of nerve cells in Hydra attenuata. Pall. Wilhelm Roux Arch. Dev. Biol. 185, 185-193 (1978) Gilula, N.B.: Gap junctions and cell communication. In: International Cell Biology (B.R. Brinkley and K.R. Porter, eds.), pp. 61-69. New York: Rockefeller University Press 1977 Gilula, N.B., Satir, P.: The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53, 494--509 (1972) Josephson, R.K., Macklin, M.: Transepithelial potentials in Hydra. Science 156, 1629-1631 (1967) Josephson, R.K., Macklin, M.: Electrical properties of the body wall of Hydra. J. Gen. Physio153, 638665 (1969) Lenhoff, H.M., Brown, R.D.: Mass culture of hydra: an improved method and its application to other aquatic invertebrates. Lab. Anim. 4, 139-154 (1970) Lentz, T.L.: Induction of supernumerary heads by isolated neurosecretory granules. Science 115, 633635 (1965) Loomis, W.F., Lenhoff, H.M.: Growth and sexual differentiation of Hydra in mass culture. J. Exp. Zool. 132, 555-573 (1956) Marcum, BA., Campbell, R.D., Romero, J.: Polarity reversal in nerve-free Hydra. Science 197, 771-772 (1977) McNutt, N.S.: Freeze-fracture techniques and applications to the structural analysis of the mammalian plasma membrane. In: Cell Surface Reviews (G. Poste and G.L. Nicolson, eds.), Vol. 3, pp. 75-126. New York: Elsevier/North Holland 1977 Passano, L.M., McCullough, C.B.: The light response and the rhythmic potentials of hydra. Proc. Natl. Acad. Sci. (USA) 48, 1376--1382 (1962) Peracchia, C., Mittler, B.S.: Fixation by means of glutaraldehyde-hydrogen peroxide reaction products. J. Cell Biol. 53, 234-238 (1972) Plattner, H.: Ciliary granule plaques: membrane-intercalated particle aggregates associated with Ca 2+binding sites in Paramecium. J. Cell Sci. 18, 257-269 (1975) Richardson, K.C., Jarett, L., Finke, E.H.: Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol, 35, 315-323 (1960) Shostak, S.: The complexity of Hydra: homeostasis, morphogenesis, controls and integration. Quart. Rev. Biol. 49, 287-310 (1974) Smith, D.S., Njogu, A.E., Cayer, M., J/irlfors, U.: Observations on freeze-fractured membranes of a trypanosome. Tissue and Cell 6, 223-241 (1974) Spurr, A.R.: A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3143 (1969) Vickerman, K.: On the surface coat and flagellar adhesion in trypanosomes. J. Cell Sci. 5,163-193 (1969) Westfall, J., Kinnamon, J. :A second sensory-motor-interneuron with neurosecretory granules in Hydra. J. Neurocytol. 7, 365-379 (1978) Westfall, J.A., Townsend, J.W.: Stereo SEM applied to the study of feeding behavior in Hydra. liT Research Institute/SEM/1976/II 563-568 (1976) Westfall, J.A., Townsend, J.W,: Scanning electron stereomicroscopy of the gastrodermis of Hydra. In: Scanning Electron Microscopy/1977, Vol. V. (1977) Wood, R.L.: The cell junctions of Hydra as viewed by freeze-fracture replication. J. Ultrastruct. Res. 58, 299-315 (1977) Wood, R.L.: The fine structure of the hypostome and mouth of Hydra. I. Scanning electron microscopy. Cell Tissue Res. 199, 307-317 (1979) Accepted January 25, 1979
Cell and Tissue Research 9 by Springer-Verlag 1979
The Fine Structure of the Hypostome and Mouth of Hydra II. Transmission Electron Microscopy Richard L. W o o d Department of Anatomy University of Southern California School of Medicine, Los Angeles, USA
Summary. The normal morphology of the hypostome and mouth of hydra were examined by transmission electron microscopy with conventional thin sections and freeze-fracture replicas. Myonemes of the hypostome are small in diameter, have gap and intermediate-type cell junctions within each epithelial layer and are associated with the opposite epithelial layer by transmesogleal processes and gap junctions. Nematocysts and sensory cells are aggregated in the circumoral region. The fine structure of adherent flagella arising from gastrodermal gland cells, and the transition region at the mouth between epidermis and gastrodermis are described in detail for the first time. The possible functional significance of the findings is discussed. Key words: H y d r a - H y p o s t o m e - Myonemes - Flagella - Freeze-fracture
As indicated in the companion paper (Wood, 1979) the hypostome of hydra has a central role in m a n y physiological activities. In the adult this region is involved with highly complex movements during capture and ingestion of prey, and during development and regeneration it is involved with the establishment of the polarity of the differentiating polyp. In order to understand better the mechanism of mouth opening, the process of engulfment and, eventually, the sequence of morphological changes in hypostome differentiation, it seemed essential to examine the fine structure of the entire hypostome of normal animals in greater detail. This paper presents the results of studies of thin sections and freeze-fracture replicas from the hypostome of normal adult hydra. Send offprint requests to: Richard L. Wood, Ph.D. Department of Anatomy University of Southern
California School of Medicine, 2025 Zonal Avenue Los Angeles, California 90033, USA Acknowledgements. The technical assistanceof Ms. AileenKuda and Mr. Watchara Pimprapaiporn and suggestions for improvingthe manuscript by Dr. Douglas Kellyare gratefullyacknowledged.Supported by grants PCM 77-14635 from the National ScienceFoundation and by funds from the LivingStructure Fund of the Department of Anatomy
0302-766X/79/0199/0319/$04.00
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Materials and Methods Cultures of Hydra littoralis and Hydra attenuata were maintained in the laboratory by the methods of Loomis and Lenhoff (1956) or Lenhoff and Brown (1970). Animals were anesthetized and fixed in most cases by the methods described in the previous paper (Wood, 1979) for routine thin sectioning, and were fixed in 0.75 ~ glutaraldehyde plus 0.75 ~ formaldehyde in 0.05 M cacodylate buffer, pH 7.4, for 1530 rain for freeze-fracture. In some instances sodium phosphate was used for buffering and either 2 ~o tannic acid or a few drops of 30 ~oH202 were added to the primary aldehyde fixatives (Peracchia and Mittler, 1972). Glutathione-treated animals were fixed without anesthetization. Specimens were embedded either in Araldite 502 (Wood, 1977) or in Spurr's low viscosity resin (Spurr, 1969). Blocks were mounted for either cross or longitudinal sectioning using a Porter-Blum IIB ultramicrotome. One-lma sections were stained with methylene-blue-Azure II (Richardson et al., 1960), mounted in immersion oil and photographed with a Leitz Ortholux microscope. Thin sections were collected on carbon-coated grids and stained with uranyl acetate/lead citrate to enhance contrast. For freeze-fracture, hypostomes of aldehyde-fixed animals were removed from the body stalk, trimmed free of tentacles under a dissecting microscope and either sliced in half longitudinally with a razor blade by hand or embedded in 3 ~ agar and chopped at 100 txm with a Sorvall TC-2 tissue chopper. Tissue slices were then glycerinated for 20-30rain each in 10~, 20~ and 30~o glycerol in 0.05 M cacodylate buffer, placed on gold alloy specimen discs and frozen in liquid Freon 22 held near its freezing point with liquid nitrogen. Fracturing and replication were accomplished in a Balzers 301 apparatus equipped with an electron gun for platinum-carbon shadowing and a film thickness monitor. Replicas were cleaned in methyl alcohol, household bleach and sulfuric acid, rinsed in distilled water and picked up on formvar-coated, 75-mesh copper grids. Thin sections and replicas were examined and photographed with a JEOL 100-C electron microscope.
Observations The h y p o s t o m e , like o t h e r b o d y regions, consists o f two epithelial layers s e p a r a t e d b y a thin m e s o g l e a ; a n i n t e r r u p t i o n in this p a t t e r n occurs at the m o u t h . I n the following account, the two epithelial layers a n d the m o u t h region are described separately.
Epidermis I n relaxed a n i m a l s the e p i d e r m i s is thin over m o s t o f the h y p o s t o m e (Fig. 1). Relatively large, e p i t h e l i o m u s c u l a r cells f o r m a n e a r l y c o n t i n u o u s layer in which n e m a t o c y t e s , n e m a t o b l a s t s , interstitial cells a n d nerve cells are intercalated. The apices o f e p i t h e l i o m u s c u l a r cells are a t t a c h e d to each o t h e r b y septate j u n c t i o n s a n d there are n u m e r o u s gap j u n c t i o n s a l o n g their lateral surfaces. W i t h the exception o f m a t u r e n e m a t o c y t e s a n d sensory cells, which are a t t a c h e d apically to epit h e l i o m u s c u l a r cells by septate junctions, n o n e o f the o t h e r cell types possess well d e v e l o p e d cell junctions. The e p i t h e l i o m u s c u l a r cells c o n t a i n v a r i o u s sized vacuoles a n d there is a layer o f irregularly s h a p e d granules u n d e r l y i n g the external surface. T h e granules m a y a p p e a r either dense o r lucent d e p e n d i n g on the m e t h o d o f fixation (Figs. 2 a n d 3). The free surface o f e p i d e r m a l cells is covered b y a cuticular layer, p r e s u m a b l y derived f r o m the c o n t e n t s o f the subsurface granules. In freeze-fracture p r e p a r a t i o n s the m e m b r a n e at the free surface o f e p i d e r m a l cells c o n t a i n s closely p a c k e d i n t r a m e m b r a n o u s particle a r r a y s o n the P fracture face a n d c o m p l e m e n t a r y a r r a y s o f pits o n the E fracture face (Figs. 4 a n d 5).
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Fig. 1.a 1 ~m parasagittal section of hypostome. The arrow indicates a nematocyst, x 380 The basal region of epitheliomuscular cells is drawn out into multiple myoid processes (myonemes) which run longitudinally (radially if viewed from above the mouth). This corresponds to the arrangement of epidermal myonemes in other body regions. Measurements were made of cross sectional diameters of 100 consecutive myonemes in a randomly selected section through each of four different body regions of a well expanded, large animal. As can be seen from the summary of these data in Table 1, the average size of the myonemes of the distal hypostome is smaller than those of the tentacles and proximal hypostome. The differences are highly significant statistically (p < 0.0005, Student's t-test). When apical hypostome and gastric region myonemes were compared there was no significant difference. Myonemes are attached to each other end-to-end, and to some extent on lateral surfaces, by intermediate-type intercellular junctions (Figs. 6 and 7). Interdigitated myonemes are also joined laterally by gap junctions (Figs. 6 and 7, arrows). In addition, small cytoplasmic processes extending across the mesoglea make gap junctional contact between epidermal and gastrodermal cells (Fig. 8, arrow). Counts of the number of transmesogleal contacts per linear extent of mesoglea 1
Unlessotherwiseindicated, all figures are from Hydra attenuata
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Fig. 2. Epidermis of hypostome of Hydra littoralis after combined aldehyde-OsO 4 fixation. C eutical layer; G subsurface granules; N nerve cell. x 7300 Fig. 3. Epidermis of hypostome of Hydra littoralis after sequential aldehyde-OsO 4 fixation. Labels as in Fig. 2. • 5500 Fig. 4. Intramembranous particle arrays on P face of epidermal cell. x 125,500 Fig. 5. Complementary pit arrays on E face of epidermal cell. x 125,500
Table 1. Comparison of myoneme sizes in different body regions
Region
Number of measurementsa
Average area in Ixm2
S.E.
Tentacles Distal hypostome Proximal hypostome Gastric region (column wall)
100 100 100 100
1.54 0.65 0.92 0.66
0.089 0.043 0.054 0.036
a For these purposes the myonemes were measured in cross sections from micrographs taken at 2,700X and enlarged to 6,750X. The assumption was made that the myonemes were square or rectangular and area was calculated as equal to average length X average width. All myonemes in a field were counted and fields were selected randomly
Fig. 6. Epidermal myonemes show intermediate junction (right) and gap junction (arrow)joining lateral surfaces. N neurite. • 47,500 Fig. 7. End-to-end junction of epidermal myonemes showing intermediate junction. Arrows indicate gap junctions, x 46,500
Fig. 8. Transmesogleal gap junction in Hydra littoralis (arrow). GS gastrodermal cell; M mesoglea; N nerve cell in epidermis, x 15,500 Figs. 9 and 10. P- and E-face images of mesogleal surface membranes of epidermal cells showing focal specialization. • 62,500 Fig. 11. 1 ~tm section showing aggregation of sensory cells (arrows) and ganglion cells near mouth
(GAN). NEM nematoblasts, x 320
TEM of Hypostome of Hydra Table
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2. Number of transmesoglealcontactsa in different body regions
Region
Distance along mesoglea Numberof Number in ~tm contacts ~rn
Tentacles Distal hypostome Proximal hypostome Gastric region
200 220 200 194
32 113 50 48
0.16 0.51 0.25 0.25
a For these data, all obvious transmesoglealprocesseswere counted even where actual contact was not observed within the section. Sections of approximately0.1 ~tm thickness were used throughout
show that such contacts are most numerous in the distal hypostome, (Table 2). The difference in number between distal and proximal hypostome is a factor of 2 and between distal hypostome and tentacles is a factor of 4. Not surprisingly, the number of contacts was the same in the gastric region as in the proximal hypostome. Epidermal myonemes in all body regions appear to have a distinctive feature when viewed by freeze-fracture. Small clusters of intramembranous particles appear on elevations on the P face of the cell membrane where it is apposed to the mesoglea (Fig. 9). A complementary depression occurs on the E face of the same membrane (Fig. 10); some such depressions display arrays of complementary pits. The significance of this intramembranous specialization is not entirely clear but it may reflect regions of firm attachment to the mesoglea. Nematocysts and nematoblasts are present in the epidermis of the hypostome, particularly in the circumoral region (Figs. 1 and 11). They occur within nematocytes that are embedded in epitheliomuscular cells. Nematocytes in the hypostome are not arranged in batteries as is characteristic for the tentacles. The freeze-fracture morphology of hypostomal nematocysts does not differ remarkably from that of tentacle nematocysts; the latter will be the subject of a separate communication. The hypostomal epidermis contains many small basally located cells. Some of these are clearly nematoblasts and interstitial cells, but most appear to be nerve cells (Figs. 2, 3, 11, 12 and 13). Neurites are represented by numerous small cell processes containing either groups ofmicrotubules or clusters of 80-250 nm vesicles, many of which have dense cores (Fig. 13). Since nerve cells of hydra have been studied extensively by other investigators (Burnett et al., 1964; Lentz, 1965; Davis et al., 1968; Bode et al., 1973; Westfall and Townsend, 1976; Epp and Tardent, 1978; Westfall and Kinnamon, 1978) the details of their fine structural morphology will not be reiterated here. It should be noted, however, that the present observations do demonstrate an aggregation of sensory cells in the circumoral region (Fig. 11). The morphology of these sensory cells appears similar to those of the tentacles as described recently by Westfall and Kinnamon (1978). An apical club-shaped cilium is present but it rests in a surface depression and does not extend above the general cell surfaces (Fig. 13).
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Fig. 12. Interstitial cell in epidermis of Hydra titwralis. E external surface; M mesoglea. • 5500 Fig. 13. Sensory cell reaching epidermal surface near mouth. SC sensory cilium; M mesoglea; N neurite with dense cored vesicles, x 5900
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Fig. 14. Longitudinal section of hypostome, mouth at top of field. G1 and G2, gland cells with two granule types; D digestive cells with microvilli borders; V phagocytic vacuoles. • 5400
Gastrodermis Gastrodermal cells of the hypostome are predominantly glandular. Most of the cells contain large secretory granules of irregular density but a few contain smaller dense granules. It is not certain whether these represent different cell types or merely different secretory phases of the same cell. Some non-glandular digestive cells are also present (Fig. 14,D). Both glandular and digestive cells possess flagella. It is clear from both thin sections and freeze-fracture replicas that flagella of the glandular cell are directed
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aborally and lie in close proximity to the luminal surfaces of the gastrodermal cells(Figs. 14, 15 and 16). A few flagella in the hypostome extend freely into the lumen; it is presumed that these arise from the digestive cells but their low frequency has made it difficult to determine their origin conclusively. Except for flagella, the glandular cell surfaces are relatively smooth, whereas digestive cell surfaces are covered with lamellar folds and elongate microvilli. The digestive cells also contain phagocytic vacuoles of various sizes. The flagella of glandular cells arise in pairs from typical basal bodies but more than one pair may arise from a single cell. The proximal region of the flagellum is constricted slightly where the basal body terminates and the axoneme commences (Fig. 16). The narrowest part of the constriction measures about 0.2 gm in diameter in thin sections. Beyond the constriction the flagellum expands slightly and the surface membrane assumes a scalloped or fluted appearance. More distally the flagellar cytoplasm expands towards the underlying cell and the axoneme remains eccentrically located along the free edge (Fig. 16). Cross sections show that fluting begins at the junction between basal body and flagellar shaft and continues onto the shaft proper (Fig. 17). There are nine flutes, each associated with a peripheral doublet by a density having a champagne glass configuration (Figs. 17 and 18) as has been described for lamellibranch cilia (Gilula and Satir, 1972). Cross sections also reveal that the more distal portion of the flagellum has wing-like lateral extensions that may be up to 0.8 l~m in total width (Fig. 19). The lateral extensions are smaller in the distal half of the flagellar shafts but the basic appearance is similar except at the extreme distal tip. Both transverse and longitudinal sections show that the expanded flagellar cytoplasm contains a condensation of filamentous material that appears to maintain an association with the peripheral doublets of the axoneme by delicate strands of dense material (Figs. 16, 19 and 20). The expanded surface of the flagellum lies in a groove along the surface of the underlying cell but the two plasma membranes remain separated by a distance of 25-30nm. The cytoplasm of the underlying cell, as well as the 25-30 nm intervening space, may also contain electron-dense material (Figs. 16 and 20). This special relationship to underlying cells occurs over the entire flagellar length, with the exception that the close apposition is lost where the flagella pass from one cell surface to the next, and at the distalmost tip. In freeze-fracture replicas the most proximal portion of the flagellum displays a 2-3 stranded neclace of P-face intramembranous particles (Figs. 21 and 22). The Fig. 15. E-faceviewof lumenalsurfaceof gastrodermalcell showingflagellarpairs runningin grooves.
x 23,200 Fig. 16. Proximal portion of adherent flagellum.B basal body; A axoneme; F filamentousmaterial connectingaxonemewithflagellarsurfaceapposedto underlyingcell.X, Y, Z, sectionlevelsfor Figures 17, 18, and 19. x 32,250 Fig. 17. Section through basal plate (X Fig. 16) of adherent flagellum. • 152,000 Fig. 18. Sectionthrough fluted region (Y Fig. 16) of adherent flagellum, x 152,000 Fig. 19. Section through shafts (Z Fig. 16) of adherent flagella.Note density of flagellar-cellsurface interspace, I. x 103,000
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Fig. 20. Longitudinal section of adherent flagellum (top) apposed to underlying cell (Hydra littoralis). Note filamentous condensation between axoneme and flagellar surface (arrows). x 98,300 Fig. 21. Proximal portion of flagellum showing necklace and fluted region, P face image, x 43,400 Fig. 22. Proximal flagellum showing P face particle clusters on membrane flutings (arrows). x 80,000 Fig. 23. Proximal flagellum E-face image of fluted region. • 52,500
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adjacent basal body region may also have an aggregation of 12.5-15 nm P-face particles. Distal to the flagellar necklace, the flagellar shaft narrows to about 0.2 I~m in diameter and then expands into a fluted region about 0.3-0.4 ~m in diameter and up to 0.6 lam long. The P-face ridges on the fluted portion have clusters of 12.5 nm particles and the interposed grooves are nearly particle-free (Figs. 21 and 22). The E face image of the fluted portion shows a few 10-12.5 nm particles in the grooves (complementary to the P face ridges) and particle-free ridges (Fig. 23). Beyond the fluted portion, the flagellar shaft is somewhat irregular in diameter; the portion housing the axoneme averages about 0.3 ~tm in width and the expanded wings range from 0.44).8 ~tm in total width. The indentations of the underlying cells, along which the flagella run, match the width of the expanded wings (Fig. 15). The shaft of the flagellum has typical 12.5nm intramembranous particles, predominantly on the P face, and some 8-10nm low profile particles (Fig. 21). Particle distribution appears to be random over most of the length of the free border of the shaft, although increased numbers of 12.5nm particles appear on some flagella proximally. The latter may be indicative of forming flagella. By contrast, the expanded portion facing the underlying cells has few 12.5 nm particles but has 8-10nm particles (Fig. 24, arrow). This pattern of particle distribution occurs on both P and E faces of the expanded wings and on P and E faces of the matching groove in the underlying cell. Absorptive cells have numerous microvilli on their lumenal surfaces and also may have several closely apposed flagella associated with these surfaces. All gastrodermal cells reaching the lumen are attached to each other by septate junctions apically and there are numerous gap junctions along lateral cell surfaces. Basally, some gastrodermal cells possess myonemes that course circumferentially. As noted above, gastrodermal myonemes extend small cytoplasmic processes into and through the mesoglea to make gap junctional contact with the myonemes of the epidermal layer. Gastrodermal myonemes do not possess the small clusters of Pface intramembranous particle arrays on their mesogleal surfaces that are characteristic of epidermal myonemes.
Mouth
The mouth region is characterized morphologically as a zone of transition between epidermis and gastrodermis. Although it can be identified in non-feeding animals, the details of the arrangement of cells in the transition zone is most readily studied in animals with open mouths. As noted by scanning electron microscopy in the preceding paper (Wood, 1979), the boundary between the two epithelial layers is marked by a depression or groove underlain by transitional cells (Fig. 25). Most of the transitional cells do not possess typical features of gastrodermal cells, such as secretion granules or flagella, nor do they contain subsurface granules or have the external cuticular layer characteristic of epidermal cells. The transitional cells are attached to each other and to adjacent epidermal and gastrodermal cells by both septate and gap junctions. Smaller, basally located nerve cells lacking obvious junctional specializations are also present near the mouth, as are neurites containing dense-core vesicles
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Fig. 24. E face of gastrodermal cell lumenal surface, L and P-face of flagellum (arrow). • 54,500 Fig. 25. Transitional area at edge of open mouth, Hydra littoralis. The flagella in the transitional groove (left) are gastrodermal in origin. C cuticular layer; E epidermal cell; G gastrodermal gland cell; T transitional cells; M mesoglea; N neurite; V phagocytic vacuoles, x 8700 Fig. 26. P face image of transitional cell external surface. Intramembrane particle aggregates (arrows) are small in size and sparse in number, x 55,600
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(Fig. 25). As noted previously there is also an aggregation of spindle-shaped sensory cells in the circumoral region. Sensory cells are attached apically to adjacent epitheliomuscular cells by septate junctions. The mesoglea is attenuated and terminates at a variable distance from the mouth edge, allowing extensive direct contact between epidermal, gastrodermal and transitional cells (Fig. 25). There are broad regions of the latter cellular contacts that lack obvious junctional specializations. Myonemes of both epidermal and gastrodermal cells become small and irregularly disposed in the area of mesoglea termination. The transitional cells do not possess recognizable myonemes. Freeze-fracture replicas show that the transitional region also exhibits a gradation in membrane morphology. Surface membranes of epidermal cells near the mouth show a reduction in the number and size of P-face particle arrays (Fig. 26) (Compare with Fig. 4) and there is a total absence of the particle arrays on some of the transitional cell surface membranes. The particle arrays have never been observed on gastrodermal cells.
Discussion
This study has provided a considerable amount of new information that adds to our understanding of the organization of the hypostome as it may relate to general functions. It also provides a basis for interpreting the results of studies now in progress on the formation of the hypostome during regeneration. The most pertinent findings will be discussed under the headings: (1) Hypostomal Movement, (2) Transitional Zone, and (3) Adherent Flagella.
Hypostomal Movement In observing the feeding activity of hydra, the complexity of coordinated movements of the hypostome and tentacles is striking. It seems obvious that there must be extensive interaction between circularly and longitudinally oriented myonemes of the two epithelial layers and mechanisms for fine control of these interactions must exist. Three morphological features of the hypostome are candidates for involvement in such fine control: (1) The hypostome has the greatest number of nerve cells of any body region. (2) The number of contacts between epidermal and gastrodermal myonemes and, concomitantly, the number of gap junctions involving cell processes from the two epithelial layers, is larger in the hypostome than in any other body region. (3) The average size ofmyonemes (crosssectional diameter) is small in the hypostome and decreases apically. Bode and coworkers (1973) have determined that over 20 % of the cells of the hypostome of Hydra attenuata are neural cells. The only other body region that has an equivalent relative number of neural cells is the basal disc. Since there is a well documented developmental gradient centered in the head region of hydra (Shostak, 1974), it has been advocated that neural cells are the source of diffusible substances involved with this gradient (Lentz, 1965). Recent evidence that nerve-free hydra can regenerate normally indicates that the developmental gradient cannot be
TEM of Hypostomeof Hydra
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maintained by neural cells alone (Marcum et al., 1977). Although the possibility that neural cells might serve as a source of morphogens has understandably excited attention, the high numbers of these cells in the hypostome suggests that they probably play an important role in more routine physiological activities as well. In fact, physiological studies have provided evidence that the major function of hydra neural cells may relate to the initiation of spontaneous contractions of the musculature (Passano and McCullough, 1962). An additional function may be involvement in fine control of localized movements. The fact that nerve-free hydra do not exhibit normal hypostomal activity but must be fed by placing food material into the enteron directly (Campbell, 1976) is consistent with such a function. The distribution of neural cells on the hypostome has not been completely elucidated. One point in question is whether or not there is a concentration of specialized sensory components in the circumoral region. It was noted in the previous paper (Wood, 1979) that cnidocils and other types of sensory hairs could not be distinguished morphologically on the hypostome in our scanning electron micrographs. However, transmission electron microscopy has confirmed that ciliated sensory cells similar to those recently described for the tentacles (Westfall and Kinnamon, 1978) do occur on the hypostome and they appear to be more numerous around the mouth. This is not surprising in view of the normal sequence of events during feeding where the mouth opens as prey organisms are drawn to the hypostomal surface by tentacular contraction. The fact that mouth opening can be induced chemically suggests that activation of the circumoral sensory cells is at least partially a chemically mediated phenomenon. A second feature of the hypostomal region that seems likely to be associated with precise localized movements is the extensive gap-junctional contact within each epithelial layer and across the mesoglea (Wood, 1977). Although there is no direct evidence that gap junctions of hydra are involved with intercellular communication, the electrical properties of the body wall in normal (Josephson and Macklin, 1967 and 1969) and nerve-free animals (Campbell et al., 1976) coupled with the mounting evidence that gap junctions serve this role in other systems 9(Gilula, 1977), support this assumption. Thus, increased numbers of gap junctions would be generally regarded as evidence indicating an increase in intercellular communication. Of particular significance in this regard is the large number of gapjunctional contacts in the hypostome between myonemes situated on opposite sides of the supporting mesoglea. This would be predicted if gap junctions were indeed involved in fine coordination of contractile activity of the two muscular layers in this region. There is scant morphological evidence that neurites traverse the mesoglea so it seems likely that such coordination must be accomplished, at least in part, by the gap junctional contacts. It is of interest, however, that the interaction between circularly and longitudinally oriented myonemes cannot simply be the induction of a comparable simultaneous activity. Coordination must involve opposing and independent activity of the two layers. That this is probably so is supported by the observation that the two layers of myonemes respond differently to urethane anesthesia (Macklin, 1976). Thus, the mechanism of transmesogleal contractile coordination is not immediately obvious, but a role for the numerous gap junctions seems likely. The third feature of hypostomal organization that may be important in fine control of movement is the size of individual epidermal myonemes. Smaller
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myonemes would be expected to result in less powerful contraction, but would also facilitate more delicate local movements if one assumes that the myonemes are capable of independent activity and that the larger numbers of neurons in the hypostome reflect increased innervation of myonemes. The present observations are consistent with this concept. Other factors such as relative lengths of each myoneme, or number of myonemes per epithelial cell, also would have an effect on fine control of movement, but quantitative evidence on these features is difficult to obtain because of sampling problems.
Transition Zone The cells of the transition zone at the mouth edge have morphological features different from typical gastrodermal and epidermal cells. Aside from representing a transition in morphology from gastrodermis to epidermis, it seems likely that the region has other functional significance. One possibility is that some of the transitional cells represent cells being lost in normal cell turnover by degeneration or sloughing. Kinetic studies of cell turnover in hydra (Campbell, 1967) have shown that cells are lost at the extremities, including the apex of the hypostome. The present observations show that phagocytic vacuoles containing cellular debris are present in some of the transitional cells. The present preparations did not show images that could be interpreted as sloughing cells, but such views could have been missed because of sampling difficulties inherent with thin sectioning techniques. A second possibility is that the transitional zone has a special sensory function. The cells that reach the surface do not appear to be degenerating but do differ in morphology from other epithelial cells in the vicinity. Although they do not appear to possess ciliary specializations, they could still be sensory in nature. Typical sensory and ganglion cells are present close by with which the transitional cells could be interacting. There is no evidence at this time that the transitional area represents a proliferative zone for nematocytes or any other cell type and the transitional cells have not been observed to be undergoing mitosis. However, this does not exclude the possibility that they may be differentiating sensory cells, a possibility that deserves further attention.
Adherent Flagella A major contribution of the present study is the elucidation of the fine structural morphology of the adherent flagellum. The close relationship of these flagella to underlying cells noted previously by scanning electron microscopy (Westfall and Townsend, 1977; Wood, 1979) is confirmed in the present study and further details are reported. The evidence that these flagella are truly adherent, and thus relatively immotile, is indirect, but taken in total perspective the evidence is fairly compelling. (1) These flagella appear to arise from only one cell type and to be directed invariably toward the base of the hypostome. (2) The expanded wings of cytoplasm characteristic of these flagella lie in grooves along the apices of gastrodermal cells, and the close apposition of the two plasma membranes is reminiscent of the relationship of flagella to cell surfaces in trypanosomes (Vickerman, 1969; Smith et al., 1974) for
TEM of Hypostomeof Hydra
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which flagellar adhesion is well documented. In the latter case, there are regularly disposed, small junctional complexes along the flagellar groove that are not present in hydra, but it has been suggested that these complexes may be involved with anchoring of intercellular components to the cell surface rather than with actual flagellar attachment (Smith et al., 1974). (3) Adherent flagella of hydra have filamentous condensations in the expanded portion facing the underlying cells, as does the subjacent cytoplasm of the latter cells. The intervening intercellular space is of constant width and also contains electron dense material. (4) Intramembranous particle distribution differs from other regions of the same membranes for both the flagellar wings and the underlying grooves. The reduction in numbers of regular sized particles (12.5 nm in this case) is reminiscent of the intramembranous appearance of intermediate-type junctions in higher organisms (McNutt, 1977). All of these features indicate a special functional relationship between these flagella and the underlying cells and are strongly suggestive of adhesion with at least partial immobilization of flagellar movement. The morphology of the proximal part of the adherent flagella is also noteworthy. As with most other flagella and cilia so far examined, a ciliary necklace occurs around an initial conical segment. This is followed by a region of prominent fluting of the membrane which extends for a variable distance onto the flagellar shaft. The flutes are precisely arranged with respect to the peripheral doublets of the axoneme and, apparently are anchored to the latter by dense strands that end within the membrane in small aggregates of 12.5 nm intramembranous particles. This configuration seems most similar to what has been described for gill cilia of clams (Gilula and Satir, 1972) but also bears some resemblance to the granule plaques of the cilia of Paramecium (Plattner, 1975). For these cilia it is suggested that intramembranous particle aggregates associated with the basal body region are involved in binding and exchange of calcium ions (Plattner, 1975), but this possibility has not been explored for hydra. The biological function of adherent flagella in the hypostome of hydra can only be speculated upon at this time. The axoneme appears normal in organization insofar as it has been examined to date. It is possible that these flagella simply serve to protect the lumenal surfaces ofgastrodermal cells by helping to hold a coating of mucus, as suggested by Westfall and Townsend (1977), but the close relationship to underlying cells demonstrated here suggests a more complex function. In trypanosomes, adherent flagella initiate undulatory movements of whole cells and it is possible that this kind of movement of lumenal surfaces of gastrodermal cells could occur in the hypostome of hydra. This possibility deserves consideration since in normal feeding behavior swallowing appears to involve an active creeping of the open hypostome around the prey organism rather than the mouth merely serving as a receptacle for material being pushed downward by the tentacles. Adherent flagella could play an important role in conjunction with myoneme contractions that implement creeping motion. This hypothesis is readily testable and experiments are being designed to study these flagella more extensively. References
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Burnett, A.L., Diehl, N.A., Diehl, F.: The nervous system of hydra. II. Control of growth and regeneration by neurosecretory cells. J. Exp. Zool. 157, 227-236 (1964) Campbell, R.D.: Tissue dynamics of steady state growth in Hydra littoralis. II. Patterns of tissue movement. J. Morph. 121, 19-28 (1967) Campbell, R.D.: Elimination of Hydra interstitial and nerve cells by means of colchicine. J. Cell. Cci. 21, 1-13 (1976) Campbell, R. D., Josephson, R,K., Schwab, W.E., Rushforth, N.B.: Excitability of nerve-free Hydra. Nature 2,62, 338-340 (1976) Davis, L., Burnett, A., Haynes, J.: Histological and ultrastructural study of the muscular and nervous systems in Hydra. II. Nervous system. J. Exp. Zool. 167, 295-332 (1968) Epp, L., Tardent, P.: The distribution of nerve cells in Hydra attenuata. Pall. Wilhelm Roux Arch. Dev. Biol. 185, 185-193 (1978) Gilula, N.B.: Gap junctions and cell communication. In: International Cell Biology (B.R. Brinkley and K.R. Porter, eds.), pp. 61-69. New York: Rockefeller University Press 1977 Gilula, N.B., Satir, P.: The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53, 494--509 (1972) Josephson, R.K., Macklin, M.: Transepithelial potentials in Hydra. Science 156, 1629-1631 (1967) Josephson, R.K., Macklin, M.: Electrical properties of the body wall of Hydra. J. Gen. Physio153, 638665 (1969) Lenhoff, H.M., Brown, R.D.: Mass culture of hydra: an improved method and its application to other aquatic invertebrates. Lab. Anim. 4, 139-154 (1970) Lentz, T.L.: Induction of supernumerary heads by isolated neurosecretory granules. Science 115, 633635 (1965) Loomis, W.F., Lenhoff, H.M.: Growth and sexual differentiation of Hydra in mass culture. J. Exp. Zool. 132, 555-573 (1956) Marcum, BA., Campbell, R.D., Romero, J.: Polarity reversal in nerve-free Hydra. Science 197, 771-772 (1977) McNutt, N.S.: Freeze-fracture techniques and applications to the structural analysis of the mammalian plasma membrane. In: Cell Surface Reviews (G. Poste and G.L. Nicolson, eds.), Vol. 3, pp. 75-126. New York: Elsevier/North Holland 1977 Passano, L.M., McCullough, C.B.: The light response and the rhythmic potentials of hydra. Proc. Natl. Acad. Sci. (USA) 48, 1376--1382 (1962) Peracchia, C., Mittler, B.S.: Fixation by means of glutaraldehyde-hydrogen peroxide reaction products. J. Cell Biol. 53, 234-238 (1972) Plattner, H.: Ciliary granule plaques: membrane-intercalated particle aggregates associated with Ca 2+binding sites in Paramecium. J. Cell Sci. 18, 257-269 (1975) Richardson, K.C., Jarett, L., Finke, E.H.: Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol, 35, 315-323 (1960) Shostak, S.: The complexity of Hydra: homeostasis, morphogenesis, controls and integration. Quart. Rev. Biol. 49, 287-310 (1974) Smith, D.S., Njogu, A.E., Cayer, M., J/irlfors, U.: Observations on freeze-fractured membranes of a trypanosome. Tissue and Cell 6, 223-241 (1974) Spurr, A.R.: A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 3143 (1969) Vickerman, K.: On the surface coat and flagellar adhesion in trypanosomes. J. Cell Sci. 5,163-193 (1969) Westfall, J., Kinnamon, J. :A second sensory-motor-interneuron with neurosecretory granules in Hydra. J. Neurocytol. 7, 365-379 (1978) Westfall, J.A., Townsend, J.W.: Stereo SEM applied to the study of feeding behavior in Hydra. liT Research Institute/SEM/1976/II 563-568 (1976) Westfall, J.A., Townsend, J.W,: Scanning electron stereomicroscopy of the gastrodermis of Hydra. In: Scanning Electron Microscopy/1977, Vol. V. (1977) Wood, R.L.: The cell junctions of Hydra as viewed by freeze-fracture replication. J. Ultrastruct. Res. 58, 299-315 (1977) Wood, R.L.: The fine structure of the hypostome and mouth of Hydra. I. Scanning electron microscopy. Cell Tissue Res. 199, 307-317 (1979) Accepted January 25, 1979
