The epitheliomuscular cell of hydra: Its fine structure, three-dimensional architecture and relation to morphogenesis

TISSUE & CELL 1978 10 (4) 629-646 Published by Longman Group Ltd. Printed in Great Britain

DAVID

L. WEST

THE EPITHELIOMUSCULAR CELL OF HYDRA: ITS FINE STRUCTURE, THREE-DIMENSIONAL ARCHITECTURE AND RELATION TO MORPHOGENESIS ABSTRACT. Ectodermal epitheliomuscular cells of Hydra attenuate were studied by transmission and scanning electron microscopy, and a three-dimensional model was constructed. These cells are cuboidal to columnar, and each cell has one muscle process arising from the basal portion of the oral-facing surface and one from the aboralsurface. Adjacent epitheliomuscular cells are joined apicolaterally by septate junctions. Numerous gap junctions occur between adjacent epitheliomuscular cells and are irregularly distributed along the lateral and basal aspects. Finger-like interdigitations and specialized folds (couplers) also occur along the basal and lateral aspects and interlock adjacent epitheliomuscular cells. In the basal portion of these cells, myofilaments are aggregated into myonemes which are oriented in the oral-aboral axis of the polyp. Myonemes dominate the cytoplasm of muscle processes. Myofilaments are also aggregated in the basal cytoplasm of the cell body when the cell body is in contact with the mesoglea but are sparse or absent when the cell body rests upon other muscle processes. Epitheliomuscular cells and associated muscle processes rest upon other processes and the mesoglea and show variations in these relations. A muscle process and associated cell may rest upon another process; the process may then extend under the preceding process and cell body. This configuration, and variations, present a woven or braided network of muscle processes which collectively form a sheet of muscle on the mesoglea. The interdigitations, couplers and gap junctions between epitheliomuscular cells and the woven network of muscle processes present a cytological basis for the observations that the ectoderm in hydra behaves as a coherent sheet along the body column.

Introduction

continuous and occurs at the tentacles, hypostome, and basal disc (Brien and Reniers-Decoen, 1949; Campbell, 1967b, 1974a). The polyp extremities continually wear away and as much as the distal fourth of each tentacle is lost daily (Campbell, 1967b). Cell loss also occurs during the periodic activity of budding which involves the separation of a portion of parental tissue with little change in cellular composition (Bode et al., 1973). Cell replacement occurs over the entire body column (Campbell, 1967a, c; Corff, 1971; David and Campbell, 1972), and the cells are in continuous movement along the column toward the sites of tissue loss. The two epithelial layers move as coherent sheets but not necessarily in register with one another (Campbell, 1967b. 1968;

THE morphology of an animal may be considered as the balance between the various morphogenetic activities of its constituent cells and tissues. One simple morphostatic form, consisting of two epithelial layers, is that of the fresh-water cnidarian, hydra, Hydra undergo perpetual growth, tissue loss and replacement, all balanced, resulting in the form of the polyp. Cell loss in hydra is

Center for Pathobiology, University of California, Irvine, California 92717. Present address : Biology Program, Sangamon State University, Springfield, Illinois 62708, U.S.A. Received 6 June 1978. Revised 9 August 1978. 629

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Shostak and Kankel, 1967). Since steadystate hydra remain relatively constant in size, cell numbers and cell composition (Bisbee, 1973; Bode et al., 1977), tissue loss is balanced by tissue growth. Many morphogenetic events in animals have been attributed to changes in cell shape which lead to the distortion of cell layers, or by the abilities of cells to undergo locomotion (Gustafson and Wolpert, 1963, 1967; Webster, 1971; Wessells et al., 1971). Localized differences in cell proliferation have also been proposed as causal factors in morphogenesis (Berrill, 1961; Burnett, 1966), but in hydra this hypothesis is not supported by experimental and histological evidence (Campbell, 1968, 1974a; Webster, 1971). During bud morphogenesis in hydra, the ectodermal epitheliomuscular cells are reoriented, and within the bud their final axial orientation is perpendicular to the original parental axis (Clarkson and Wolpert, 1967; Otto, 1977). Hydra consisting only of epithelial cells (i.e. lacking nerve, interstitial and nematoblast cells) also undergo morphogenesis including budding (Diehl and Burnett, 1965; Campbell, 1976; Marcum and Campbell, 1978; Otto, 1977). The mechanical Forces which cause morphogenetic activity must then reside in the epithelial cells, or in conjunction with the mesoglea and/or hydrostatic pressure of the gastric cavity. Since epitheliomuscular cells are implicated in morphogenetic activities, consideration should be given to the architecture of these cells, with reference to their abilities to alter cell shape and contact and to undergo locomotion. Campbell (1968, 1974b) considers cell movement and adhesion important factors in determining form in hydra and suggests that the muscle processes, septate junctions, and vertical contractile elements provide a cytological basis for the morphogenetic abilities of these cells. These cytological elements have been studied by light and electron microscopy (Hess, 1961; Wood, 1959,1961,1977; Chapman, 1974). However, no adequate three-dimensional model for this cell in hydra is available. The present study was undertaken to review the fine structure and to determine the three-dimensional morphology of the epitheliomuscular cell and investigate the relationships between these components and the morphogenetic activities attributed to this cell,

Materials and Methods

Stock Hydra attenuata were maintained in modified ‘M’ solution (containing NaCl instead of NaHCOa) according to the general methods of Lenhoff and Brown (1970). Cultures were fed Artemia nauplii daily, and the culture medium was changed after each feeding. Animals used for electron microscopy were starved approximately 24 hr before fixing. Transmission electron microscopy

Animals were placed in 10ml of culture medium in Petri plates (6 cm diameter) and relaxed by gradual warming in a 50°C oven, about 3-5 min, until they did not contract when lightly stimulated. Animals were quickly flooded with 10 ml of 0.1 M phosphate buffer (pH 7.4) containing 4% glutaraldehyde and 2 % formaldehyde which was prepared from paraformaldehyde (Karnovsky, 1965). Specimens were subsequently transferred to freshly prepared fixative (2% glutaraldehyde; 1% formaldehyde, made from paraformaIdehyde; 0.05 M phosphate buffer, pH 7.4; one-half strength modified ‘M’ solution). Unrelaxed animals were prepared in the same manner as described above but were maintained at room temperature before quickly flooding with fixative. All tissues were fixed for 1 hr at room temperature and subsequently washed for 30 min in 0.05 M buffer, pH 7.4, containing 2% sucrose. Tissues were post-fixed for 1 hr at room temperature with 2% 0~04 in 0.05 M phosphate buffer, pH 7.4, dehydrated through a graded series of acetones (10 min each), and embedded in an Epon 812-Araldite 502 mixture (Anderson and Ellis, 1965). Thin sections were cut with a diamond knife and stained with aqueous uranyl acetate followed by lead citrate. Sections were examined and photographed with a Siemens 1A electron microscope. There are no apparent morphological differences between unrelaxed animals and those relaxed by gradual warming. For localization of carbohydrates, the periodic acid-thiocarbohydrazide-silver proteinate technique (Pearse, 1972) was employed. Cell reconstruction Cross and longitudinal sections were cut 8001000 A thick (estimated from interference color) from the mid-gastric region. Serial

EI’LTHELIOMUSCULAR

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sections were collected, four to six sections per grid, on Formvar coated, single slot (I x 2 mm) grids. A total of 60-90 grids (approximately 40-45 wrn of tissue) were collected from each of one longitudinally sectioned and two cross-sectioned animals. Photographs were taken of an identifiable area from each grid and were approximately 0.5-l pm apart. Occasionally, photographs were taken of successive sections. A cell was selected and its outline traced onto clear plastic sheets from the photomicrographs. The tracings were arranged in sequence and the morphology of the cell determined. Three cells were reconstructed in this fashion. One of these cells was also reconstructed in a wax model. The outlines of this cell were cut from sheets of paraffin wax (7-8 mm thick) and all sections were assembled in sequence. Spacers (about 8 mm thick) were inserted between wax sections to adjust for section spacing. Scanning electron microscopy

Unrelaxed hydra or hydra relaxed in 2% urethan in culture medium were fixed for 3-4 hr in the primary fixative listed above. Tissues were dehydrated in a graded series of ethanols, exchanged in a graded series of absolute ethanol-Freon 113 and critical point dried with Freon 13. Specimens were mounted on aluminum studs and coated with gold-palladium. For examination of muscle processes relaxed animals were transferred to a few drops of maceration fluid (David, 1973). After 30-60 set in this fluid the ectoderm would loosen from the mesoglea. Animals were quickly transferred into primary fixative and the ectoderm was carefully peeled back using jewelers forceps while observing under a dissecting microscope. Large areas of ectoderm were peeled as a coherent sheet and left attached to the body column to facilitate subsequent handling. Tissues were fixed for 30 min in primary fixative and subsequently treated with the glutaraldehyde-tannic acid-osmium tetroxide technique of Sweney and Shapiro (1977). Tissues were critical point dried and mounted as described above. For internal features of epitheliomuscular cells, animals were cut into two or three pieces at various angles to the long axis of the body with razor blades while in primary fixative, or, following critical point drying, pieces were broken off with forceps. Specimens were examined with

an Hitachi HISCAN operated at accelerating potentials of 5, 10 or 15 kV. Photographs were taken with Polaroid P/N 55 film.

Observations Morphology and fine structure In surface view in relaxed animals, epitheliomuscular cells are essentially fusiform in outline (Fig. 1) with the long axis oriented in the oral-aboral axis of the polyp. The cells of a bud are also fusiform but oriented in the long axis of the bud. At the bud-parent junction, however, the cells are more circular in outline (Fig. 2). In the contracted state, the polyp surface is highly irregular (Fig. 3), and the apical surfaces of epitheliomuscular cells are variously folded (Fig. 4). Occasionally, the lateral walls are folded across the basal portion of the cell. Epitheliomuscular cells, when dissociated by David’s (1973) maceration technique, are columnar to cuboidal and present irregular surface features (Fig. 5). These cells have muscle process extending from the basal region, and in the ectoderm, muscle processes are oriented in the oral-aboral axis of the polyp; each cell has a muscle process extending from the oral-facing surface and one from the aboral-facing surface (Fig. 5). In maceration preparations muscle processes show some diversity in length and configuration. In some instances, a process may extend only 5 pm or so from one end of the cell, while at the other end the process may extend up to 20 pm from the cell border. In other cells, muscle processes may extend equidistantly from the two ends of the cell and measure 40-50 pm in total length. The basal portion and associated muscle processes of epitheliomuscular cells rest upon the mesoglea and muscle processes from adjacent cells. Collectively they form a sheet of muscle on the mesoglea (Figs. 6, 7). In sectioned material, the major portion of an epitheliomuscular cell volume consists of a large central vacuole (Fig. 8). The cytoplasm is generally restricted to the periphery of the cell and is l-3 pm in thickness (Fig. 8). Irregularly distributed cytoplasmic strands often span the central vacuole. Smaller vacuoles are scattered throughout the cytoplasm, some of which are continuous with the central vacuole. The nucleus, lo-14 pm

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wide, is irregular in outline and surrounded by a layer, 0.5-l pm thick, of cytoplasm (Fig. 8). The nucleus is enclosed by a layer of cytoplasm, or suspended by strands, on the periphery of the cell. The surface of hydra is covered by a layer of material, the periderm, which is composed of two layers of filamentous material (Figs. 9, 10). The outermost layer (20&400 nm thick) is a loose aggregate of filaments which are generally oriented perpendicular to the cell surface and abut upon the inner layer (Fig. 10). The inner layer (20-25 nm thick) is composed of a compact aggregate of filaments which appear to be oriented parallel to the cell surface. The inner layer of

periderm is separated from the cell membrane by a variable distance ranging from 5 to 150 nm, and the space between them occasionally contains a flocculent or granular material. The material of the periderm reacts positively to the periodic acid-thiocarbohydrazide-silver proteinate (PATCSP) technique indicating the presence of carbohydrates (Fig. 10) which are mucopolysaccharides since the periderm also stains with alcian blue. The apical plasmalemma of epitheliomuscular cells also reacts intensely to PATCSP, indicating the presence of carbohydrates, perhaps a glycocalyx. Within the cell, the apical layer of cytoplasm contains free ribosomes, scattered

Abbrt wiations used in figures C CE co D E EM z IA IN JG JI JN

Cnidocil Connections between endo- and ectodermal epithelial cells ‘Coupler’ Dense-cored vesicle Rough endoplasmic reticulum Epitheliomuscular cell Mesogleal fibers Golgi complex Electron-dense inclusions Interdigitations of adjacent cells Gap junction Fascial intermediate junction Synaptic junction

JS ME MF MP N NC

NE P S T vc VI

Septate junction Mesoglea Myofilaments Muscle process Nucleus Nematocyte Nerve Periderm Spirochaete within mesoglea Microtubules Central vacuole Vacuole in apical layer of cytoplasm

Fig. 1. Scanning electron micrograph of the body column in relaxed Hydra attenuata; arrow indicates oral-aboral axis of animal; note ridge outlining cell boundaries. x 700. Fig. 2. Surface view of relaxed animal showing bud-parent junction; note circular outline of epitheliomuscular cells (*) in this region; arrow indicates oral axis of bud. x 860. Fig. 3. Surface view of contracted

polyp.

x 700.

Fig. 4. Scanning electron micrograph of a longitudinally note folding of apical surface (*) due to polyp contraction.

cut epitheliomuscular x 5400.

Fig. 5. Epitheliomuscular cell in Hydra attenuafa. Nomarski differential-interference optics.) x 1500.

(Maceration

Fig. 6. Scanning electron micrograph showing muscle sheet formed muscular cells; arrow indicates oral-aboral axis. x 1500. Fig. 7. Tangential above the mesoglea;

cell;

preparation; by epithelio-

section through muscle processes of epitheliomuscular arrow indicates oral-aboral axis. x 10,000.

cells just

Fig. 8. Cross-section of epitheliomuscular cell from mid-gastric region; myofilaments are aggregated in regions of the cell body which are in contact with the mesoglea (*). (Section forms part of wax model.) x 9500.

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mitochondria, a few profiles of rough endoplasmic reticulum, and microtubules. This region also contains membrane-bounded, electron-dense inclusions (Figs. 8-10). These inclusions are circular to oval in outline, measuring 1-2 pm in length, and are generally situated near the plasmalemma. They are rich in carbohydrates which are mucopolysaccharides since they stain with alcian blue and PATCSP. The limiting membrane reacts intensely with PATCSP (Fig. 10). The similarity between the material in these inclusions and the material of the periderm suggests that they give rise to the periderm, but fusion of inclusions with the plasmalemma, in an exocytotic manner. or indications of fusion were never observed throughout this study. However, empty vesicles which have the same dimensions as inclusions occur in the apical cytoplasm (Fig. 8). Also, some inclusions appear as vesicles containing varying amounts of material. However, the mechanism of material release remains obscure. The cytoplasmic layer on the lateral limits of epitheliomuscular cells generally contains more kinds of organelles than in the apical and basal portions, but no particular organelle is restricted in distribution. This cytoplasmic region (Fig. 11) has long profiles of rough endoplasmic reticulum which, in favorable sections, appear continuous from the basal region, along the lateral aspect, into the apical region. Cisternae of the endoplasmic reticulum are dilated, 43-45 nm wide, and contain a moderately electrondense material (Fig. 11). Groups of Golgi lamellae are scattered throughout the cytoplasm, and each group consists of 8-12 flattened saccules. Cisternae of the Golgi complex contain an electron-dense material, and numerous vesicles are associated with the tips of the saccules (Fig. II). Microtubules are scattered throughout the cytoplasm (Figs. 1I, I2), and in the lateral region they generally occur in loose bundles oriented in the apicobasal axis (Fig. 11). These microrubules have been observed extending from the basal region to about two-thirds of the cell height, and short segments of microtubules occur in the upper lateral portion of the cell. They may extend, unbroken, the entire height of the cell, but this was never confirmed. Multivesicular bodies and residual bodies are randomly distributed and generally occur in the lateral cytoplasm. Residual

bodies are occasionally quite large, up to 12 pm in width. The basal cytoplasm of epitheliomuscular cells contains two sizes of myofilaments, thick and thin, with thin filaments predominant (Fig. 13). Thick filaments are 14-l 5 nm in diameter, and thin filaments measure 5-6 nm in diameter. Both sizes of filaments are oriented in the long (oral-aboral) axis of the cell (Figs. 7, 8, 12, 13) and are randomly distributed with respect to each other. Myofilaments are aggregated into bundles forming myonemes which dominate the cytoplasm of muscle processes (Figs. 7, 12). They also occur within the basal cytoplasm of the cell body. However, in areas in which the cell body rests upon other muscle processes myofilaments are sparse or absent (Figs. 8, l2), but when the cell body region is in contact with the mesoglea, myofilaments form myonemes (Figs. 8, 12). Myofilaments abut upon the plasmalemma in a few places forming hemidesmosome-like structures. Bundles of microtubules extend into the muscle processes and pass along the process just above the myoneme. Mitochondria and free ribosomes are scattered throughout the basal cytoplasm and muscle processes (Fig. 12). Dense-cored vesicles also occur within these regions and are situated near the plasmalemma (Figs. 7, 12, 20). These vesicles are 80-140 nm in diameter, with the core measuring 30-50 nm in diameter. They are similar in appearance to the dense-cored vesicles found in nerve ceils but are smaller in diameter (see Fig. 20). Muscle processes do not appear to anastomose (Fig. 14) but have irregular margins (Figs. 7, 14) and, occasionally, fairly long lateral or terminal finger-like projectionc. Cell velatiomhips Epitheliomuscular cells are joined to adjacent epitheliomuscular cells by extensive septate junctions (Figs. 8, 9). These junctions form a belt around the apicolateral margin of the cells and are similar in morphology to those of Hydra (= Pclnlatohydra) oli,uctis described by Wood (1959, 1961). At the junction of adjacent cells, the two cell borders are slightly evaginated forming a small cone projecting above the cell surface in sectioned material (Fig. 9). This configuration is visualized in surface view as a small ridge outlining cell boundaries (Figs. 1, 2). This

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ridge, however, is obliterated in contracted animals (Fig. 3). Along the sides of adjacent epitheliomuscular cells, many finger-like extensions interdigitate with adjoining cells (Figs. 7, 8). In addition to septate junctions and interdigitations, specialized foldings also occur between cells and interlock them (Figs. 8, 15). These folds, ‘couplers’, can be visualized in a fashion as couplings between railroad cars. These couplers, along with interdigitations, also occur in the basal region and along muscle processes (Figs. 7, 15). Numerous gap junctions occur between epitheliomuscular cells and are irregularly distributed (Figs. 1l-13, 16). Adjacent muscle processes are joined by fascial intermediate junctions (Wood, 1977) which resemble fasciae adherentes found in the intercalated disc of cardiac muscle. These junctions (Figs. 7, 13, 15) only occur between muscle processes of different cells and are irregularly distributed along the lengths of processes. Muscle processes which are in contact with the mesoglea send out small projections basally into the mesoglea and

make contact with endodermal cells across the mesoglea (Figs. 6, 8, 13). These connections have gap junctions between them (Fig. 16) and show various profiles in micrographs, ranging in outline from simple endto-end abutments to ‘ball and socket’-like configurations (Figs. 13, 16). Muscle processes show great variation in their relationships to other muscle processes and to the mesoglea. In some instances, a muscle process, or a lateral portion of it, may extend on top of another muscle process for the major part of its length before coming in contact with the mesoglea. Other processes may have several areas of contact with the mesoglea. However, all epitheliomuscular cells make contact with the mesoglea at some point. The lateral margins of muscle processes are also irregular and form interdigitations with adjacent processes (Fig. 7). Muscle processes which are in contact with the mesoglea have other processes and epitheliomuscular cell bodies on top of them (Figs. 8, 12); these processes may then extend on top of preceding processes. This configuration,

Fig. 9. Cross-section of apical cytoplasmic region muscular cells; note ridge formed by small evaginations Fig. 10. Periderm and electron-dense thiocarbohydrazide-silver proteinate.)

inclusions x 42,000.

at junction of two epithelioat cell borders. x 14,500.

within apical region. (Periodic

Fig. 11. Cross-section through lateral cytoplasmic region; folded across the basal region due to contraction. x 19,000.

the lateral

acid-

region

is

Fig. 12. Longitudinal section showing muscle processes and basal region of epitheliomuscular cells: note absence of myoneme in basal region when cell body rests upon muscle processes (*). x 16,500. Fig. 13. Cross-section

through

muscle processes

Fig. 14. Scanning electron micrograph longer period in maceration fluid (compare Fig. 15. Longitudinal x 21,000.

section through

Fig. 16. Gap junction between to left of figure). x 139,000.

and mesoglea.

x 29,000.

of muscle processes; Figs. 17, 18). x 1300.

basolateral

endodermal

preparation

region of epitheliomuscular

and ectodermal

connection

from cell.

(ectoderm

Fig. 17. Scanning electron micrograph of muscle processes showing relationships of adjacent processes; note process of cell (*) extends on top of adjacent processes (arrows). x 3400. Fig. 18. Scanning electron micrograph of muscle processes; appearance (arrows) of adjacent processes. x 4200.

note woven or braided

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and various other combinations, appear to form a woven or braided network of muscle processes (Figs. 17, 18). Interstitial cells, nerve cells, and nests of nematoblasts are situated between epitheliomuscular cells (Fig. 19). These cells rest upon muscle processes or the basal portions of epitheliomuscular cells and, in this study, have never been observed in contact with the mesoglea. Nerve cell bodies are generally restricted to the region just above muscle processes and send out many axons which extend between and along muscle processes (Fig. 20). Axons also extended apically between epitheliomuscular cells. Interstitial cells and nematoblasts, in places of occurrence, fill the space between epitheliomuscular cells, from just above the muscle processes to the lower limits of septate junctions. Cell membranes of surrounding epitheliomuscular cells are closely apposed to interstitial cells and nematoblasts, but cell junctions were never observed between epitheliomuscular cells and these cells. However, synaptic junctions occur between epitheliomuscular and nerve cells (Fig. 20). Mature nematocytes are individually mounted in pockets in the apical surface of epitheliomuscular cells along the body column (Fig. 1). Nematocytes are enclosed by the apical surface of epitheliomuscular cells, with septate junctions between them (Fig. 21). The cnidocil projects a short distance beyond the body surface. Mesogleu The mesoglea is an acellular matrix which separates the ectodermal and endodermal epithelial layers and is similar in many respects to the basement membrane of other metazoans. It is collagen-like in chemical composition and is made up of filaments embedded in an amorphous matrix (Figs. 13, 23). These filaments react positively to PATCSP (Fig. 22). The matrix also reacts moderately to PATCSP (Fig. 22). Mesogleal filaments are randomly distributed within the matrix and abut upon the plasmalemma of muscle processes and epitheliomuscular cell bodies in a few places. Spirochaetes inhabit the mesoglea of H. atlenuata used in this study (Fig. 22). Bacterial inhabitants of the mesoglea seem to be widespread in some species of cultured hydra (Hausman, 1973).

In surface view, after removal of the ectoderm, the mesoglea presents an irregular surface which reflects the conformation of the muscle processes and is perforated by numerous holes (Fig. 23) which accommodate the connections between the two epithelial layers. In preparations which were left in maceration fluid for longer periods of time (l-2 min), the mesoglea is smooth and has no holes, suggesting that the epithelial connections are inserted through the mesoglea and when the ectoderm is removed, before the mesoglea is stabilized by the fixative, the mesoglea smooths out and the holes disappear. Three-dimensional morphology The wax reconstruction of the epitheliomuscular cell from the mid-gastric region presents an irregular outline, but the general shape is cuboidal (Figs. 24-27). The apical surface (Fig. 25) is indented for a short distance, beginning about one-third of the way back from the oral-facing surface. This indentation is due to the contracted condition of the polyp (see Figs. 3,4). This cell is about 12 pm high, 17 Frn long, excluding muscle processes, and 11 pm wide. Along the lateral aspect, nematoblasts are situated between this cell and its neighbours, on both sides, in the oral facing half of the cell length (Figs. 24, 25). The irregularities along the sides of the model show the numerous indentations and projections between adjacent epitheliomuscular cells. The basal portion of the cell rests upon other muscle processes throughout its length and only a small portion, about 5 pm long and 2-3 pm wide, makes contact with the mesoglea (Figs. 8, 26). The two muscle processes are on top of other processes throughout the length which was followed. Unfortunately, the entire length of the muscle processes could not be followed with certainty. The oral-facing muscle process arises in the midline of the cell and extends under the preceding cell. The aboral facing muscle process is off center (Fig. 27) and sandwiched between other processes. Along the basal portion of the cell body, a few lateral projections extend over adjacent muscle processes and make contact with the mesoglea at one point (Fig. 26). Fig. 28 presents the three-dimensional morphology of epitheliomuscular cells from the mid-gastric region of

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H. attenuata.

This model is stylized from three cells which were reconstructed. Discussion

Within recent years, many advances have been made toward understanding the underlying mechanisms of morphogenesis. Cell growth, proliferation, migration, and changes in cell contact are reported to be causal factors in morphogenesis (Gustafson and Wolpert, 1963, 1967; Campbell, 1974a, b). However, in hydra, and apparently hydrozoans in general (Braverman, 1974), it is clear that cell proliferation does not play a major role in morphogenesis (Campbell, 1967a, c, 1968; Shostak et al., 1965). Further, cell division may not be necessary for morphogenesis at all, since hydra in which DNA synthesis and mitoses have been blocked still undergo regeneration and morphogenesis (Webster, 1971). It seems that the relation between morphogenesis and cell proliferation is that cells divide as a response to morphogenesis and growth (Campbell, 1974b). Current

evidence is in support of the hypothesis that cell or tissue movement and changes in cell adhesion are primary factors involved in hydra morphogenesis. Tissue movement in hydra has been reported by many investigators using a variety of markers (Campbell, 1967b; Shostak and Globus, 1966; Shostak and Kankel, 1967). The epithelial layers move along the body column toward the oral and aboral ends, and the layers move as coherent sheets. The two layers, however, may move at different rates, with one marked layer moving past the other (Tripp, 1928; Brien and ReniersDecoen, 1949; Campbell, 1967b; Shostak et al., 1965). Results from the present study provide a cytological basis for the observations that the ectoderm moves as a coherent sheet. The extensive interdigitations and couplers between epitheliomuscular cells suggest that they are firmly interlocked, and the woven network of muscle processes, with fascial intermediate junctions between them, further suggests that independent cell movements are infrequent, if they occur at all.

Fig. 19. Scanning electron micrograph nematoblasts (*) between epitheliomuscular

of ectodermal cells. x 1800.

Fig. 20. Cross-section through muscle nerve cell and muscle processes. x 16,000.

processes

Fig. 21. Tangential mounted nematocyte. Fig. 22. Cross-section proteinate.) x 27,500. Fig. 23. Scanning removal of ectoderm.

section through x 26,500. through electron x 2800.

apical

mesoglea. micrograph

region

(Periodic showing

epithelium;

showing

note nests of

relationship

of epitheliomuscular

between cell with

acid-thiocarbohydrazide-silver surface

of the mesoglea

after

Fig. 24. Side view of wax model reconstruction of epitheliomuscular cell; oral facing axis indicated by arrow; nematoblasts occurred along lateral region (*). Approx. x 5000. Fig. 25. Apical-lateral view of wax model of epitheliomuscular cell; oral-facing axis indicated by arrow; the apical surface is indented (arrowhead) due to contraction of animal (cf. Fig. 4); nematoblasts occurred along lateral surface (*). Approx. x 5000. Fig. 26. Wax model of epitheliomuscular cell viewed from oral-facing end. The cell rested on muscle processes and came in contact with the mesoglea in the central region of the cell body (*). Approx. x 5000. Fig. 27. Aboral surface view of wax model of epitheliomuscular cell; arrow indicates oral-facing axis; note the muscle process of the aboral-facing surface arises laterally to centre line of cell. Approx. x 5000.

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Fig. 28. Diagrammatic representation of epitheliomuscular region of H,wira attmmta: stylized from three reconstructed

In addition, epitheliomuscular cells and their associated muscle processes have numerous gap junctions between them, and also between the endodermal and ectodermal connections. These gap junctions appear to have increased adhesive properties since they do not cleave in a conventional freezefracture manner (Wood, 1977). The gap junctions in hydra, which may also be responsible for the electrical coordination between the two epithelial layers (Josephson and Macklin, 1967; Hufnagel and KassSimon, 1976), are of the ‘B’ type, having intramembranous particles adhering to the B fracture face rather than the A face as in all reported vertebrate gap junctions (Larsen, 1977; Wood, 1977). Wood (1977) reported that a high proportion of gap junctions in hydra have cytoplasmic fragments partially or totally covering the junction following freeze-fracture which indicates that the forces within the membrane and in the intermembranous spaces are more stable than is

cel( from the mid-gastric ceils.

typical for gap junctions. Filshie and Flower (I 977) also found greater adhesive properties in hydra gap junctions. They reported that these junctions did not separate after glycerination, although septate junctions separated, but that considerable distortion occurred. and they suggested that there may be a localized bonding function of the gap junctions. Thus, the interlocked nature ot epithelial cells and the numerous gap junctions which may have an adhesive function indicate that the ectodermal layer is functionally a coherent sheet of cells which behaves as a unit along the body column. Tissue and cell movements in hydra are associated with morphogenesis, and morphostasis is either a result of or a requirement of cell or tissue movement. Campbell (1974aj considers that movement of the epithelial layers may be due to either active movement or to passive displacement. Active movement of the epithelial layers is suggested to occur in two cases (Campbell, 1974a). One case is

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during column expansion when the epithelial layers move axially but slip past one another, and the second case is during tentacle and bud formation (Campbell, 1974a; Otto, 1977). Results from the present study reveal some morphological restrictions that epitheliomuscular cells must overcome if they actively migrate. The interlocked nature of epitheliomuscular cells and the woven or braided network of muscle processes suggest that a high degree of coordination must exist if active movement is to occur. The presence of gap junctions between epitheliomuscular cells and adjacent muscle processes and fascial intermediate junctions between muscle processes are suggestive of a coordinating pathway, but they also suggest that the sheet of ectoderm must move as a unit. Campbell (1974a) suggests an alternative hypothesis for active movement of the epithelial layers. This suggestion is that the axial movement of tissue regions away from one another is due to tissue expansion by cell proliferation and growth. The cell cycle time for epithelial cells is approximately three days (David and Campbell, 1972), and the tissue expands about one-third of the column length in 24 hr. Epithelial cells also appear to have a variable premitotic phase (Ga) which suggests that the tissue may have a rapid division response to stimuli, e.g. grafting or head removal (David and Campbell, 1972). Thus, cell proliferation and growth of the ectoderm may account for tissue movements. Considerations of active movement must also include the morphological basis for migratory abilities. The myofilaments within epithelial cells and, as a substratum, the mesoglea provide a basis for these cells to undergo movement. However, within the endodermal cells the orientation of the myofilaments is circular which is perpendicular to the direction of axial movement. The mesoglea has been suggested to act as a substratum for epithelial cell movement. However, Hausman and Burnett (1971), using 3H-proline, reported rapid (within 6 hr) incorporation of label into the mesoglea. They also reported label in the basal portions of digestive and epitheliomuscular cells. These observations indicate that epitheliomuscular and digestive cells synthesize mesoglea and that the mesoglea undergoes continuous synthesis and turnover. From studies on the structure and development of

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skin basal lamina in the fish Fundulus, Nadol et al. (1969) suggested that the basement membrane is synthesized at least in part by the overlying epithelial cells and that growth, both radial and longitudinal, is accompanied by synthesis of basal lamina and included either sliding of the layers or increase in the collagen fibril length at the end points. From similarities in the basal lamina of other metazoans and the mesoglea in hydra, growth and tissue expansion may also occur in the same relative fashion. Thus, growth and tissue expansion in hydra does not require active tissue movement using the mesoglea as a substratum. The abilities of cells to alter their areas of cell contact, which is either due to or a result of changes in cell shape, have been also implicated as factors in morphogenesis. Vertical contractile elements and septate junctions in hydra epitheliomuscular cells have been suggested to be involved in altered cell shape and areas of contact (Campbell, 1974b). Mueller (1950) reported that the muscle processes of epitheliomuscular cells appear to anastomose and that the myonemes extend from the base of the cell up into the cell body forming vertical myonemes. From the present study, the muscle processes do not appear to anastomose and the epitheliomuscular cells of Hydra attenuata lack vertical contractile elements. However, epitheliomuscular cells have well-developed bundles of microtubules which may function as a cytoskeleton and also have abilities to apply forces for cell deformation. The central vacuole may also provide additional support. The central vacuole of the cells probably functions in an osmoregulatory capacity, as suggested by Benos et al. (1977), but it may also provide a deformable but uncompressable cytoskeleton. Septate junctions have been considered to be responsible for strong epithelial coherence in hydra and also in controlling the flow of external environmental components in the fashion of a ‘leaky tight junction’ (Filshie and Flower, 1977; Benos et al., 1977). However, strongepithelial adhesion may also involve gap junctions (see above). These components of epithelial muscular cells provide a cytological basis for the morphogenetic activities residing in altered cell shape and cell contact. The foregoing considerations primarily outline questions in understanding hydra

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CELL

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morphogenesis and do not concretely answer them. However, the present findings are in agreement with Campbell’s (1974a) suggestions that active cell movement may not be involved in hydra morphogenesis, but rather morphogenesis may be viewed as a change in morphological boundaries which involve altered cell shapes and contact in conjunction with cellular differentiation. Investigations into the roles of the endodermal epithelium in morphogenesis, which has not yet been studied, are needed. Further studies into the adhesive nature of gap junctions may provide information as to the involvement of these junctions in altered cell shape through changes in cell contact. Future work along these lines may provide greater understanding

into the cellular morphogenesis.

mechanisms

involved

in

Acknowledgement Many thanks to Drs R. D. Campbell and B. A. Marcum for enlightening discussions and for reading the manuscript. Thanks to T. Novak, also for suggestion of the term ‘coupler’, and N. Wanek for their many helpful suggestions. This study was supported by Grant No. HD 07029, awarded by the National Institutes of Health, DHEW, by Grant No. PCM 77-00276, awarded by the National Science Foundation, and by Grant No. IN-120, awarded to the University of California, Irvine, by the American Cancer Society.

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