Observations on the Structure of Hydra as seen with the Electron and Light Microscopes

Although the two-layered anatomy of hydra is well known, the structural units of which it is composed are so small that use of the light microscope has never fully elucidated their arrangement. The far greater resolution afforded by the electron microscope enables some new observations to be made on the fine structure of this animal, and provides further information about the nature and distribution of the mesogloea, of the muscle-fibres, and of various kinds of cells composing the ectoderm and endoderm.

A large, colourless variety of Pelmatohydra oligactis was employed in the electron microscope study. Animals were fixed for io min to i h in Dalton’s fixative (Dalton and Felix, 1955), a solution which contains 1% OsO₄, 1% K₂Cr₂O₇ at pH 7-2, and 0-85% NaCl. Pieces of hydra were also fixed in buffered 1 % OsO₄ in Ringer’s solution for similar lengths of time. The best results were obtained after short fixation in Dalton’s fluid, and most of the electron micrographs are taken from material treated in this way.

Certain animals were placed directly in the fixative and contracted immediately. These were preserved in a contracted state. Others, which were first anaesthetized with |% chloral hydrate, remained relaxed and were neither greatly contracted nor extended when fixed. Some of the anaesthetized hydra were divided into parts (hypostome, body, pedal disk) before fixing.

After fixation, the specimens were washed in distilled water, placed in 70% alcohol, dehydrated, and then embedded in a partially polymerized mixture of one part of methyl methacrylate to three of butyl methacrylate. This was polymerized at 45° C with benzoyl peroxide as the catalyst. Ultrathin sections (about 300 Â thick) were cut with a modified Minot rotary microtome equipped with a glass knife, or with the Servall Porter-Blum microtome. Sections were taken in transverse, longitudinal, sagittal, and oblique planes. Most of the observations were made on transverse and longitudinal sections of the body, these being the specimens whose orientation and appearance were simplest to interpret. The sections were mounted on copper mesh grids, which were inserted into a Philips EMroo or an RCA-EMU type electron microscope, without removing the plastic. Negatives were exposed at a magnification of 1,000 to 6,000 times and enlarged photographically to the desired size. The final magnifications are approximate.

In order to assist the recognition of structures seen with the electron microscope, control sections were examined regularly under phase contrast. Ordinary histological preparations of white and brown P. oligactis were also of help in orientating the electron micrographs.

Frozen-dried sections of a brown variety of P. oligactis have been studied with the light microscope. After freezing-drying (see Bell, 1956), specimens were placed in methyl benzoate with 1% cellodin; the normal procedure for double embedding was followed (Pantin, 1948). Section ribbons were flattened over Baker’s formaldehyde-calcium (1944), on which they were left floating overnight at room temperature for post-fixation of the tissues. After two or three changes of distilled water, the ribbons were floated on to albumenized

Hess, Cohen, and Robson—The Structure of Hydra 317 slides, left to dry overnight, and stained by Mallory’s trichrome method (Pantin, 1948). Although post-fixation may cause sections to shrink slightly, they become easier to handle and stain more clearly. It may be noted that freezing-drying, like osmium fixation, does not produce specimens uniformly free from artifact, and that certain regions are much better preserved than others.

It should be borne in mind that all the observations recorded here have been made on fixed material. While the high magnification of the electron microscope allows fine structure to be described far more accurately than is possible with the light microscope, the relation of many structures occurring in fixed material to those found in living hydra has not yet been determined.

Epithelio-muscular cells. Certain cells of the ectoderm possess large oval nuclei of an even granular texture (fig. 1, A, nuc). Their cytoplasm contains a large variety of organelles and inclusions, among which may be found small vacuoles bounded by membranes with apposed granules, structures showing folds extending a short distance into the interior of the organelle and which may be identified as mitochondria of moderate size (fig. 1, A, mit), and complex bodies consisting of adjacent smooth membranes, vesicles, and vacuoles. There are several other inclusions, usually seen as electron-dense bodies, such as, for example, a frequently occurring ellipsoidal structure which is distinguished from mitochondria by being smaller and much denser, and without internal folds (fig. 5, F, org). Another body, although only moderately dense, is also darker than the surrounding cytoplasm (fig. 1, A, org), and is round in outline with a folded or crumpled membrane. Large and small granules, within which further structure may be detected, are seen at the centre. This type of inclusion or organelle, while varying slightly, often occurs in endodermal digestive cells as well (p. 322). A granular material adhering to the outer surface of the ectodermal cells forms a thin coat over the hydra (figs. 1, A; 5, F, surf)-

Within some of the cells may be seen closely packed bundles of fine fibrils arranged in parallel and running in the general direction of the column axis (fig. i, A, mf). The fibrils are usually accumulated at the base of the cell (compare fig. 2, c, ect), but as in fig. 1, A, some may extend upwards above the nucleus. The appearance and disposition of the bundles of fibrils indicate that they correspond to the myofibrils of histologists (see von Gelei, 1924), and the cells in which they are found are therefore epithelio-muscular cells. A detailed consideration of the structure and orientation of these intracellular fibrils further confirms their identity (see p. 322).

Several authors have reported the presence also of supporting or skeletal fibres in the epithelio-muscular cells of hydra, especially in the region of the foot and gonads. They are believed to provide stiffness and elasticity, although Mueller (1950) has suggested that some of them are in fact muscular. Now the ‘supporting fibres’ figured by von Gelei (1924) pass from the celLbody into the basal extensions of the epithelio-muscular cell and run alongside the muscle-fibres (compare fig. 6, B). They would thus correspond closely in arrangement to the fibrils which extend into the ectodermal cells as just described. We find that the fine structure of these fibrils is identical with that of the myofibrils at the base of the epithelio-muscular cells. ‘Supporting fibres’ have not usually been described as being present in the endodermal cells, and correspondingly we find that myofibrils in the endoderm are restricted to the base of the cells. Our observations, therefore, tend to confirm Mueller’s suggestion that many ‘supporting fibres’ form part of the muscular system.

Mueller (1950) agrees with von Gelei (1924) and other authors, however, in believing that a distinct class of intracellular supporting fibres does exist, but our observations so far provide no evidence for this. It must be remembered that other investigators have used different methods from those in the present study, and that under the light microscope fine fibrillar structures are near the limit of resolution and difficult to interpret with certainty.

Cnidoblasts and developing nematocysts. Another kind of ectodermal cell occurs in groups, which are sometimes found next to the mesogloea. Each cell contains a large round body which is limited by a relatively thick homogeneous border, and shows dark particles scattered throughout the interior (fig. 4, A, nem). In other cases, these organelles may be even larger, and sections reveal that they are undoubtedly developing nematocysts. The structures in fig. 4, A are therefore immature nematocysts at various stages of development, and the cells containing them are cnidoblasts.

The nucleus of each cell may be displaced by the developing nematocyst. Within the nucleus a very dense organelle showing small clear patches represents the nucleolus. It apparently does not have a thread-like or vesicular structure and is usually found towards the centre of the nucleus (fig. 4, A, n, nuc). Other electron-dense particles are also seen, scattered irregularly within the nuclear membrane.

In these cells numerous vacuoles of a moderate size are dispersed throughout the cytoplasm and intercommunicate with each other, often giving the appearance of channels. The vacuoles generally appear to be empty and are bounded by membranes. At high magnification, numerous fine granules are seen in and around the membranes, and the cytoplasm between two membranes may also appear granular. In some sections the cytoplasm immediately surrounding a nematocyst rudiment is particularly dense and shows a lamel-lated structure (fig. 4, A, lam). Bodies are also present whose internal folds and structure resemble those of the mitochondria seen in other cells, and they may therefore be identified as such (fig. 4, A, mit).

Although only one nematocyst occurs in each cnidoblast, sections of a group of developing cells show that the cytoplasm of adjacent cnidoblasts is apparently continuous at certain points along their borders (fig. 4, A, con). If this appearance is not due to fixation artifact, it may indicate that cells in a group of cnidoblasts are in syncytial relation to each other. Such a syncytial arrangement of cnidoblasts containing immature nematocysts might ensure that the batteries of nematocysts in the tentacles developed in a co-ordinated manner. The orderly pattern of mature nematocyst types has been studied by von Gelei (1927) and Semai van Gansen (1951).

An electron microscope study of hydra nematocysts has been made by Semai (1954a), who used whole mounts of discharged capsules. Four kinds of nematocysts are found. The parts characteristic of the stenotele, or penetrant type, are the capsule, leading into the shaft or basal portion of the butt and carrying projecting stylets, the distal part of the butt or conical piece, which may sometimes carry spines, and the elongated filament or tube, which extends from the conical piece and may either carry rows of spines (Hyman, 1940) or be devoid of armature (Semai, 1954a).

In sections through the capsule of a mature undischarged stenotele within its cnidoblast (fig. 3, B), the coiled and elongated filament (t) is found embedded in an electron-dense homogeneous matrix, which is presumably fluid coagulated within the nematocyst capsule. The filament is a narrow tube with homogeneous walls and a central lumen: it appears to be empty and to lack spines. The conical part of the butt is seen in figs. 3, B (è) and 3, D as two serrated structures on whose inner sides numerous dark overlapping spines are attached. Above these in fig. 3, B are the large stylets (s), within the basal part of the butt. The attachment of the butt to the capsule wall cannot be seen in these sections, but in fig. 3, D the distal end of the butt extends as the beginning of the nematocyst thread, which has been cut through.

Sections through capsules of another type of nematocyst also show structures composed of overlapping layers of electron-dense material (fig. 3, c, sp). They apparently represent sections through the barbed thread of a holo-trichous isorhiza or large glutinant nematocyst. These barbs appear similar in structure to the spines on the butt of stenoteles.

The appearance of the tube in sections of undischarged nematocyst capsules shows that the filament is already present before the nematocyst discharges and that it is not formed by extrusion of fluid or magma as suggested by Kepner and his colleagues (1951).

Groups of small, rounded cells also occur in the ectoderm (fig. i, c). They are numerous in some areas and absent from others, and appear to be the interstitial cells.

The nucleoplasm of these cells is very light, but it contains granules and an extensive network of dark substance, which may represent nucleolar material. In some nuclei this dense network may surround islands of light nucleoplasm. The cytoplasm is not very dense, and contains granules, mitochondria with internal folds, and round vacuoles of varying sizes (fig. 1, c, vac) to which granules may be apposed. In some sections a complex of smooth membranes and vacuoles is seen.

If the interstitial cells give rise to cnidoblasts (fig. 4, A; see Hyman, 1940), they must undergo remarkable changes in organization, especially in the case of the nucleolus and of the cytoplasmic vacuoles.

Groups of secretory cells also occur in the ectoderm of the foot region. Large vacuoles, each of which usually contains a dense ellipsoidal granule, are found towards the periphery of each cell (fig. 5, A, vac, g). There is considerable space between a granule and its vacuole, probably due to shrinkage during preparation of the tissue. The cytoplasm between vacuoles contains mitochondria. The outermost cell membrane is thick and finely granular, and is covered by an external layer of dark amorphous material (fig. 5, A, al).

These cells are apparently ectodermal gland cells of the pedal disk, which are the only kind found in that region and are characteristically filled with coarse granules (Hyman, 1940). Their appearance in frozen-dried material is seen in fig. 6, E, which shows part of a vertical section of the foot (compare fig. 5, A). The peripheral granules are seen again in tangential section in fig. 6, F. The amorphous substance seen in the electron micrographs is probably the adhesive material which cements hydra to the substratum and which is secreted by these gland-cells.

In some electron microscope sections the secretory granules are found outside the foot of the hydra as if they had just been extruded (fig. 5, A) : they may rest in a depression in the outer cell-membrane or in the amorphous layer, and give the impression that they have just passed out of the cells and are still clinging to the surface. The extrusion of granules may nevertheless be an artifact, as it is possible that they were forced out of the cell mechanically during preparation of the tissue.

The mesogloea, which lies between the ectoderm and endoderm (figs. 2, A, B, c; 3, A; 5, E, mes), is non-cellular and usually appears light, although in some sections it is fairly dark. It is usually granular, and the granules may be close together or fairly scattered. In some preparations they seem to be resting on a background of extremely fine longitudinally orientated threads, and it is possible that some of the granules in the mesogloea represent cut fibrils. Other oblique sections reveal very fine threads which appear to be present throughout the mesogloea and give it a fibrillar texture (fig. 5, E, mes).

In frozen-dried preparations also the mesogloea sometimes appears to be fibrous in nature, although when poorly fixed it appears reticulate. If true fibres are present, this is a further likeness between the mesogloea of hydra and that of other coelenterates (see Chapman, 1953). The varying appearance of the mesogloea probably depends on the state of contraction in which the hydra is fixed.

The mesogloea appears to be bounded by two basement membranes, which separate it from the ectoderm and endoderm respectively. These membranes are not always easy to discern but may be seen in fig. 2, c (doub). They will be discussed further in considering the relation of the muscle-fibres and meso-gloea (p. 324). The mesogloea also shows holes filled with a very light substance (figs. 2, A, c). As will be explained below, these represent oblique sections of cytoplasmic roots, which are extensions of the cytoplasm surrounding the muscle-fibres and penetrate the mesogloea (Semai, 1952).

The endodermal cells of hydra vary in structure and appearance according to the region of the animal as well as to the time since the last meal. Two main types of cell are concerned with the digestion and absorption of food: the gland cell, which pours secretion into the lumen to break down the food and makes its products available for intracellular and perhaps extracellular digestion, and the digestive cell, which absorbs the food products. Both kinds of cell have been observed with the electron microscope.

Gland-cells

Some of the endodermal cells are almost completely filled with large vacuoles, and can be identified as ‘cellules spumeuses’ or foamy cells (fig. 4, c). Semai (19546) describes them as the most numerous kind of glandcell, filled with polygonal vacuoles of finely granular content. Their appearances in the light and electron microscopes are very similar and they are thus relatively easy to identify.

Electron micrographs of foamy cells show that the vacuoles appear to be filled with granules, or with a thread-like material, or a mixture of both (fig. 4, c, vac), according to their plane of section. At the base of the cell the vacuoles are smaller than those at the periphery and contain a denser granular material. Small mitochondria with internal folds occur infrequently in the tenuous cytoplasm between the peripheral vacuoles, but they become more numerous towards the base of the cell.

Semai (19546) has described the way in which foamy cells discharge at the moment the hydra ingests prey, and then undergo a recharging cycle. Fig. 4, c appears to represent part of a discharging cell. Its vacuoles are similar to those of a resting cell, but they may have altered in size as there seems to be more cytoplasm between them. The mitochondria, of moderate size and with short internal folds, are now easier to discern (fig. 4, c, mit). Structures consisting of overlapping or lamellated double membranes may also occur between the vacuoles of a discharging cell, either alone or near mitochondria (fig. 4, c, lam).

The discharged material of the foamy gland cells consists not only of vacuoles and their contents but also of portions of the cytoplasm with its mitochondria and lamellated membranes (fig. 4, c). This type of secretion differs from that of the ectodermal gland-cells of the foot, where only secretory granules formed in the cytoplasmic vacuoles leave the cell.

We have not yet observed the presence of muscle-fibres in these cells, nor seen their attachment to the mesogloea.

Certain other endodermal cells are more variable in structure and probably represent the digestive cells. These are known to undergo profound changes in shape and organization during the digestion of a meal (Semai, 19546). Fig. 4, B shows part of one of these cells filled with relatively small vacuoles (vac) : since digestive cells become vacuolated at the beginning of a meal (Semai, 19546), the state of this cell probably corresponds to an early stage in the digestive process. The peripheral cytoplasm extends into the lumen of the gut as numerous villous processes (fig. 4, B, vp), which are covered with external granules, and are a constant feature of these cells during digestion. In addition the digestive cells, like the foamy gland-cells, each possess two flagella (figs. 1, B ; 4, B ; 5, B “,flag), as described in the next section. Myofibrils are found in the basal cytoplasm of some digestive cells and are considered further on p. 323.

Generally speaking, the cytoplasm of the digestive cells is light (fig. 1, B) and contains vacuoles of varying sizes, with which granules and mitochrondria may be associated. Several other organelles or inclusions may be present. Of these, a round body, whose folded membrane encloses a fairly light substance with complex internal granules, much resembles a similar structure found in ectodermal cells (p. 317, fig. 1, A, org).

There is again a complex of membranes ássociated with vacuoles. Several kinds of inclusions in large vacuoles, smaller vacuoles associated with granules, and dark bodies of variable size whose density is approximately that of lipid material, are among the structures which probably represent stages in the digestion of food products. Similar inclusions have been described by Semai (19546) and they vary according to the time after a meal and the kind of food ingested.

The endodermal cells possess flagella which have been said to number from one to five, with two the most frequent estimate (McConnell, 1931 ; Mueller, 1950; Semai, 19546). In our preparations the flagella of both foamy and digestive cells constantly occur in groups of two (figs. 1, B; 5, B, flag). Sections of the flagella show the nine peripheral and two central longitudinal filaments which characterize the cilia and flagella of other animals (Fawcett and Porter, 1954). In addition there is a structureless, relatively thick surrounding membrane, which appears to be closely connected to the flagellum but often becomes detached (figs. 1, B; 4, B; 5, B, C). This thick membrane differentiates the flagella of hydra from those of associated organisms. The numerous cilia shown in fig. 5, D, for example, belong to aprotozoon situated on a hydra, and lack the thick sheath which surrounds each hydra flagellum. Part of the protozoon is included in the section.

The muscular system

Since the organization of the muscular system in hydra is as yet incompletely known and it is of considerable functional importance, relevant observations made in this study are discussed together below.

It has been seen that longitudinal sections of the ectoderm show closely packed bundles of fine fibrils, which are orientated longitudinally at the base of the cells and rest on the mesogloea (p. 317; fig. 1, A). The fibrils are spaced evenly and are not cross-striated. Their bundles form a single-layered network extending over most of the animal. In slightly oblique transverse sections the bundles of fibrils are seen to form a parallel series above the mesogloea (fig. 2, c, ect). Since the majority of observers find that the ectodermal muscle-fibres of hydra are also longitudinally orientated, and that they occupy the same positions as the bundles of fibrils we have observed, there is strong indication that the two are identical.

The myofibrils are thus found within epithelio-muscular cells, and they extend right into the long cytoplasmic processes forming the base of each cell (see fig. 6, B). Longitudinal sections sometimes show the muscle-fibres surrounded by basal cytoplasm (fig. 2, A, ect, bas), but more often the cytoplasm is so thin that the muscle-fibres seem to rest directly on the limiting membrane of the mesogloea (fig. 3, A, ect). It is interesting that the muscle-fibres are not separated from the cytoplasm by a special membrane, although when the cytoplasm in the basal extensions of the cell becomes very thin, the cellmembrane may appear to belong to the muscle-fibre (fig. 3, A). Mitochondria are often found just outside the myofibrils, and they may then bulge from the surface of the muscle-fibre (figs. 2, A, B; 3, A, mit).

The muscle-fibres are closely applied to the mesogloea and seem either to push into it themselves (figs. 2, B, c, ect, mes), or to be anchored by small pseudopodial processes which penetrate the mesogloea (see mes in figs. 2, A, B, c; 3 A). These appear to be cytoplasmic attachment roots provided by the bases of the epithelio-muscular cells, as described by Semai (1952). Longitudinal sections also show points of contact between the muscle-fibres (fig. 2, B,J). Although the membranes of adjacent fibres are intimately connected they remain distinct and the sarcoplasm and fibrils of neighbouring cells do not fuse.

The bases of digestive cells resting on the mesogloea also contain very fine, closely packed fibrils (figs. 2, A, B; 3, A, end). They are more difficult to discern than the ectodermal myofibrils owing perhaps to the particular state of contraction of the fixed hydra, but their position and structure indicate that they are of a similar nature and they therefore represent endodermal myofibrils. They are orientated at right angles to those of the ectoderm and form a transverse muscular layer: thus when the ectodermal muscle is cut longitudinally, endodermal muscle is cut in cross-section, and conversely.

All the digestive cells which contain muscle-fibres appear to rest upon the mesogloea. As in the ectoderm, the muscle-containing bases of the cells are in contact with each other, but their respective membranes remain independent, as do their sarcoplasm and fibrils. There is little cytoplasm surrounding the muscle-fibres, however, as they are usually close together and almost fill the base of the cell. While the muscle-fibres are closely connected to the mesogloea, they are smaller and penetrate it less deeply than those of the ectoderm, and cytoplasmic roots of attachment are fewer and less robust than those of the epitheliomuscular cells.

Frozen-dried sections of anaesthetized hydra stained by Mallory’s trichrome method show structures which have been seen by previous authors and also during the present electron microscope work. The muscle-fibres in particular are very clear. They stain red, as do the muscle-fibres of other coelenterates when coloured by Mallory’s method. Transverse sections show that the fibres, situated within the cytoplasm of the epithelial cells, occupy the positions indicated by electron micrographs. Fig. 6, A illustrates this feature in the ectoderm of a contracted specimen in which the muscle-fibres are especially evident. Their position within a single musculo-epithelial cell may be confirmed by comparing this photograph with fig. 6, B, showing an ectodermal cell obtained by the Hertwigs’ osmic-acetic maceration technique (1879). The muscle-fibre lies in the epithelial cytoplasm at the base of the cell, just above the mesogloea, as all recent workers using maceration methods have been able to demonstrate (Goodrich, 1942; Mueller, 1950; Semai, 1952). This has been shown in the electron micrographs (figs. 2, A, B, C; 3 A), as described above.

In contrast to the red muscle-fibres the mesogloea stains blue with Mallory’s method, and in this respect it resembles the mesogloea of anemones and medusae (Chapman, 1953). The different staining properties of muscle-fibres and mesogloea are illustrated by figs. 6, c, D, which show the same Mallory preparation photographed through blue and red filters. Fig. 6, D shows that the mesogloea forms a distinct layer between the ectoderm and endoderm (see also figs. 2, A, B, c; 3, A; 5, E). It varies in thickness with the state of contraction of the hydra and is best developed in the foot region of this species (Pelmatohydra oligactis)’, Holmes (1950) found a similar distribution in Chlorhydra viridissima.

As seen above (p. 323), the electron micrographs show that the muscle-fibres are intimately inserted on to the mesogloea, but that they are separated from it by membranes. Oblique sections in particular show that at least two membranes are present: as well as a membrane limiting the cytoplasm which surrounds each muscle-fibre (p. 323), there is a less clearly defined basement membrane belonging to the mesogloea (fig. 2, c, doub). It has long been known, however, that the muscle-fibres on each side of the mesogloea form two systems of open networks, which are orientated longitudinally, in the ectodermal plane, and transversely, in the endoderm (Mueller, 1950). Between adjacent muscle-fibres, undifferentiated epithelial cytoplasm is therefore in contact with the mesogloea, and other types of cell such as cnidoblasts, or digestive cells without muscle-fibres, may also occupy this position.

It has been suggested by Holmes (1950) that the muscle-fibres represent specializations of the mesogloeal substance and he refers to them as ‘meso-gloeal fibres’. The evidence which has been presented above makes it unlikely that the muscular system of hydra originates from a fluid mesogloea by ‘crystallizing out’ along lines of force, as has been claimed by Holmes. All types of preparation we have studied show that the muscle-fibres occur within epitheliomuscular cells or digestive cells, and that they belong to these cells and not to the mesogloea. It is difficult also to reconcile Holmes’s theory with the observation that distinct membranes are present between the muscle-fibred and mesogloea.

Holmes’s explanation (1950) of the organization of the muscular system of hydra, therefore, seems untenable. The problems which it raises, however, as to the nature and origin of the muscle-fibres and mesogloea still remain unsolved. It is to be hoped that future work with the electron microscope will be able to throw light on the much-discussed functional morphology of this small animal.

This paper was written while one of us (A. H.) was on leave of absence at the Anatomy School of Cambridge University, and another of us (E. A. R.) held a D.S.I.R. research assistantship under Dr. C. F. A. Pantin, F.R.S., in the Department of Zoology of Cambridge University. The authors wish to thank Dr. C. F. A. Pantin for his unstinted help and advice in the preparation and production of this paper. The authors are also grateful to Mr. Charles E. Houck, Jr., of the Department of Anatomy of Emory University, Georgia, for his co-operation and technical aid in the production of most of the electron micrographs.

All the electron micrographs show sections of hydra, and in these the line represents 1 μ. Magnifications are approximate.