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AMER. ZOOL., 14:523-535 (1974). Cell Movements in Hydra RICHARD D. CAMPBELL Department of Developmental and Cell Biology, University of California, Irvine, California 92664 SYNOPSIS. All cells in hydra undergo continuous and systematic locomotion across the polyp body. Different patterns and mechanisms of movement are exhibited by the various cell types. Passive displacement is one of the most conspicuous forms of cell movement: The epithelial cells of the body column and tentacles move centrifugally due to the expansive growth of the tissues. Another factor in the passive displacement of epithelial cells involves the propagation of morphological characters across the tissue, rather than actual movement of cells or tissues per se. True cell locomotion, where cells move relative to neighboring tissue by means of self-propulsion, is also apparently exhibited by epithelial cells in certain situations, notably at sites where morphogenetic changes are taking place (e.g., bud and tentacle bases) . Epithelial cells always migrate as sheets of cells, never individually. In contrast, ncmatocytes and interstitial cells undergo active migration individually. Nematocyte migration is strongly polarized, very possibly by the mat of epithelial cell muscular processes along which they move. Interstitial cell migration appears to occur under many conditions, but not nearly as commonly as was classically supposed. Finally it appears that interstitial cells and nematoblasts move in bulk through the intercellular spaces in some situations. This movement is probably due to epithelial cell activities and is used to redistribute cells located in the interstitial spaces. The cells and tissues of hydra are in continuous flux. The polyp undergoes perpetual growth and tissue loss, coupled by balanced cell renewal patterns involving all cell types. During these processes some cells migrate individually; others are pulled in sheets. The intricacies of these movements are becoming more and more apparent as experimental and histological studies progress. So far, however, the different types of cell movement occurring in hydra have been studied by different investigators and have not been well correlated with one another. This paper summarizes our understanding of the various forms of cell movements in hydra and classifies and analyzes them in both mechanism and occurrence. CELL AND TISSUE RENEWAL The occurrence of cell locomotion is related to discrepancies between the patterns of cell origin and destination. Cells arise Supported by NIH Research Career Development Award 5-KO4-CM42595 and NSF Research Grant GB 29284. by either mitotic proliferation (epithelial cells and interstitial cells) or by differentiation from interstitial cells (nematocytes and nerves) (Campbell, 1967a; David and Campbell, 1972; Davis, 1974). Gland cells divide mitotically, but it is not known to what extent, if any, they also arise by differentiation of interstitial cells. In all cell types mitotic proliferation and differentiation are widely distributed throughout the hydra body column (Fig. 1). There is no local zone of growth or cell production in the body; although it was once widely held that growth was restricted to the subhypostomal region (Brien and ReniersDecoen, 1949; Burnett, 1961), this was based on a false deduction and has now been widely found to be untrue (Campbell, 1967&; Webster, 1971). The most notable exceptions to the almost uniform distribution of cell production are that nerve cell production appears to be highest in the vicinity of the hypostome, developing bud, or site or regeneration (Schaller, 1970) and that there is little or no cell production in the tentacles, hypostome tip, and region near the basal disc. 523 524 RICHARD D. CAMPBELL Ectodermal epithelial cells o Endodermal epithelial cells . X <D o- N> Ectodermal interstitial cells "5 -o o — ' •°"""O^No 2 Endodermal gland cells I ^— o -o—— I O Position Along Hydra Column FIG. 1. Distribution of cell proliferation along the body column in Hydra littoralis. The ordinate represents the mitotic index, averaged over measurements taken throughout the day and night, of the four cell types considered: ectodermal epithelial cells, endodermal epithelial cells, ectodermal interstitial cells (including all cells in the interstitial spaces of the ectoderm), and endodermal gland and mucous cells. The abscissa indicates the body region. No mitotic activity occurs in the tentacles. (Modified from Campbell, 1967a) . CELL MOVEMENTS IN HYDRA Cell loss occurs primarily at the budding region and at the body extremities (hypostome, tentacles, and basal disc) (Campbell, 19676). Budding involves segregation of a large area of parent tissue with little modification in its cellular composition (Bode et al., 1973). Thus, all cell types are lost in the proportions in which they occur in column tissue. The polyp extremities continually wear by tissue atrophy (Brien and Reniers-Decoen, 1949), and these regions are rather specialized in cellular composition: most cells in the tentacles are nematocytes; as the distal fourth of each tentacle is lost daily (Campbell, 19676), many thousands of nematocytes (Bode et al., 1973) are shed. Hypostome and basal disc tissues are comprised mainly of epithelial, gland and nerve cells. Thus, cell formation occurs in the body column, and almost all cell loss occurs at the budding region or at the body extremities with the nematocyte loss being particularly rapid and restricted in tentacle tips. CELL MOVEMENTS The intricate, interweaving renewal patterns of the various cell types demand, or result from, several types of cell locomotion. Some cell types may display several distinct types of movement. The forms of cell translocation from one part of the body to another may be classified in categories of Passive Displacement, Active Movement (both cell and tissue), and Passive Movement. Passive displacement: The apparent displacement of cells relative to the morphology of hydra, but without cells moving relative to neighboring cells or structures Hydra tissue grows continuously. This expansion results in all tissue regions moving away from each other. Since cell and tissue loss, localized at the top and bottom of the body column, maintain constant animal size, tissue continually moves from the central body regions peripherally to the sites of cell and tissue loss. This is one 525 of the first types of tissue movement to be mapped out in hydra (Tripp, 1928). There is no a priori reason to assume that any active type of cell migration underlies these centrifugal column movements, although several instances of cell locomotory activity have been found in certain cases (see below). Rather, the tissue simply expands. Yet, there has been the greatest confusion concerning the mechanics of these column displacements. When the idea of a localized subhypostomal growth region was maintained (e.g., Brien and Reniers-Decoen, 1949; Burnett, 1961), one spoke of growth as forcing or pushing tissue proximally and distally, thus mechanically creating the column tissue movements. These descriptions are conceptually unsound. Actually, there are two components to the column tissue displacements and different mechanisms underly them. One component is the movement of tissue regions away from one another. This is due to tissue expansion by growth. The second component is the displacement of tissue regions relative to morphological boundaries. This component should be viewed not as a tissue movement phenomenon, but rather as migration of the morphology across the stationary (or expanding) tissue. The budding zone, situated near the base of the column, continually migrates upwards into the column by means of new buds always arising above older buds (Brien and Reniers-Decoen, 1949). The basal disc migrates upward along the stalk since at any time the cells at its upper edge are in the process of becoming base cells. That is, the cellular morphogenetic states associated with being basal disc cells are continually propagated upwards into naive stalk tissue. This gives the false impression that cells are moving downwards into the base; actually the base is moving upwards across the tissue. The result is similar in the two cases, but the underlying mechanism is not. A similar situation occurs at the tentacle bases; the column cells adjacent to a tentacle base are always becoming new tentacle base tissue, thus elongating the tentacles. This process has the appearance of 526 RICHARD D. CAMPBELL cells migrating outwards into tentacles. Actually, tentacle organization is propagating inwards across the tissue. While both interpretations yield equivalent patterns, the concept of centripetal morphology migration is probably closer to the mechanisms involved. Column tissue expansion and centripetal morphology migration are generally balanced, leading to steady state development. This balance is independent of growth rate. Tentacle tissue turnover rates have been measured and found to be relatively independent of feeding rates (Dunne and Campbell, unpublished). Basal disc cell loss is probably also constant. Budding, however, is facultative; it occurs only when growth is in excess of peripheral tissue loss and budding then occurs at a rate commensurate with the excess growth. Thus, tissue growth and loss are almost always balanced. They are unbalanced when feeding rates are too low to balance the constant peripheral tissue loss, and the hydra diminishes in size (Tripp, 1928). They are also unbalanced in the well-fed individuals of mutant hydra which are unable to bud (Lenhoff, 1965; Moore and Campbell, 1973a); in this case the hydra continuously grow larger to giant proportions. The combination of centrifugal tissue expansion and counterbalancing centripetal movement of morphological regions across this tissue give the appearance that cells are migrating away from the central column region. Thus, the most obvious and well-mapped tissue displacements in hydra need not involve any active cellular migration at all. Locomotion: Cells or tissues moving relative to neighboring cells or tissue structures, due to migratory behavior of the cells involved Active tissue (epithelial) migrations. There are two prominent cases where hydra epithelial cells apparently actively migrate. These are (i) during certain col- ECTODERM ENDODERM I BUDDING ZONE l0 ° 0 DAYS FIG. 2. Ectodermal and cndodermal column tissue displacements in Hydra viridis. The column positions of tissue markers (ordinate) are plotted as a function of time (abscissa) . Colloidal carbon was used to mark the ectoderm and symbiotic algae were used to mark the endoderm. The tissue behavior of four (fl-d) animals is shown. In a-c, the ectoderm is displaced distally relative to the endo- derm; depending on the position of the markers initially, the ectoderm and endoderm are displaced distally or proximally. In d, both tissues move precisely in register proximally; this latter behavior was always observed wihen the markers were originally in the lower two-thirds of the gastric column. (From Campbell, 1973.) CELL MOVEMENTS IN HYDRA 527 umn expansions, where ectoderm and endoderm move relative to one another, and (ii) during formation of buds and tentacles. In these cases, migration involves the epithelia as sheets rather than single cells. The locomotory structure is probably the muscular process of the epithelial cell. In column tissue displacements, the two tissue layers may or may not move in register. In Hydra viridis the tissues move together in lower column regions, but within the upper quarter of the column they move at different rates (Fig. 2). In two other cases where ectoderm and endoderm column differential displacements were looked for they were also found (Shostak et al., 1965; Campbell, 1967c) so probably they are of widespread occurrence. However, the particular patterns of differential movement differ with species and probably with different culture and growth conditions. Shostak et al. (1965) demonstrated the ability of an isolated epithelial sheet to migrate, using the mesoglea as a substratum. Interestingly, they found that such migrations occurred in only one direction, as might be expected from tissues of such a highly polarized organism. Their experiments indicate that the mesoglea probably acts as a substratum for this locomotion. The other situations which apparently involve epithelial cell migration occur at the sites of tentacle and bud formation. FIG. 3. Orientation of muscular processes at the margin o£ a developing bud of Hydra viridis. This longitudinal section of a young hud extends from the junction with the parent (at extreme left) to about 1/6 of the length of the bud. The bud tip is toward the right. The section is perpendicular to the parent column axis. The ectoderm is at the top and the mesolamella runs across the lower portion of the illustration. At far left, the three vertical arrows indicate three ectodermal epilheliomuscular cell processes which arc perpendicular to the plane nf Ihe micrograph; Ihese are parallel to the parent body axis, as ate all ecUxlerinal muscular processes in the parent. At the extreme right, the muscular processes lie within the plane of the micrograph. Thus, in this marginal region of the bud the epithelial cells rotate as adjacent parent tissue becomes incorporated into the bud. The thickening of the mesolamella seen at the right, where numerous cellular processes extend into and through it, is typical of this site of cell rotation. The three oblique arrows in the right center indicate three nearly mature nematoblasts (the nematocyst is visible only in the lowest one) which have moved from their site of differentiation higher in the epillielinn to the muscular layer lining the inesolaniella. x 1,900. 528 RICHARD D. CAMPBELL Shostak and Kankel (1967) showed that the position of a bud's base moves slightly along the column in H. viridis as the bud matures. This could well involve epithelial cell migrations, although the pattern is complex and its mechanism has not been studied. Also, in both the tentacles and buds, new epithelial cells continually add on to the outgrowth's base all around it. In the ectoderm the epithelio-muscular cell processes are all oriented strictly axially along the tentacles and body column (Mueller, 1950), and in buds of all stages (Campbell, unpublished). Therefore, the epithelial cells entering tentacles or buds from their sides must change their orientations by 90°. This change in orientation occurs near the base of the outgrowth and involves a gradual rotation. Probably it is accomplished by the ectodermal cells migrating slightly across the mesoglea; those cells above and below the outgrowth can migrate directly on without rotation; those cells to the side migrate in a curved path which results in the rotation. A region of epithelial cell rotation at the margin of a bud is shown in Figure 3. Analogous migrations could occur in the endoderm, although there the pattern of muscular processes is much more complex and has not been worked out in the vicinity of bud and tentacle bases. In epithelial cell migrations, one probably should consider the muscular process as the organelle responsible for the movement. The muscular process has the appearance of a locomotory organelle. It also exhibits the behavior necessary: it is contractile and highly mobile. During tissue healing the muscular process is the first cell extension which crosses and closes a tissue gap (Bibb, 1971), and it may be just as active in normal tissue. Epithelial cell migration seems to use the mesoglea as a substratum (Shostak et al., 1965) (there is no other obvious substratum), and the muscular process is the only portion of the cell or tissue contacting the mesoglea. Another possible mechanism (Campbell, 1968) of epithelial cell migrations is altered cell-cell contact relations which are thought to cause morphogenetic movements in embryos (Gustafson and Wolpert, 1962; Burnside and Jacobson, 1968). Active single cell migrations. Nematocytes: Nematocytes originate in the body column of hydra and many subsequently migrate to the tentacles where they become mounted for use. Nematocyte migration occurs entirely within the ectoderm (Campbell, 1967c). The migration begins by the nearly-mature nematoblasts settling toward the base of the ectodermal epithelium (Fig. 2; 3 oblique arrows). This migration occurs just at the time that their synchronous developmental clusters, or nest, disaggregates. Then they independently migrate distally towards and into the tentacles. Although migrating nematocytes are close to the mesoglea, they generally do not touch it but instead glide along the upper surface of the epithelial cell muscular processes, which form a continuous, axially oriented corrugated mat. It is reasonable to suspect that this highly sculptured substratum guides and orients nematocyte movements, much as tissue cultured cells may show "contact guidance" along much finer surface features (Weiss, 1961). However, this question has not been pursued experimentally, and different nematocyte types behave differently (Herlands, 1972) indicating considerable complexity in the control of nematocyte migration. The migrating desmoneme nematocyte is a strongly polarized cell. The leading edge consists of a pseudopod, behind which is the cell nucleus. The nematocyst capsule trails at the back of the cell, with the cnidocil end pointing backwards (Fig. 4). This orientation appears to be usual for nematocytes (Giinzl, 1971; Rahat and Campbell, 1973). In the body column of hydra the nematocytes migrate distally up the column from their site of origin; this is demonstrated by grafting isotopically labeled tissue into unlabeled columns and analyzing with radioautography the proximal and distal distributions of labeled nematocytes after varying times (Fig. 5). CELL MOVEMENTS IN HYDRA FIG. 4. Nematocytes migrating at the base of the ectodermal epithelium towards the tentacles in Hydra attenuata. This is a surface view of a whole, fixed hydra, at the focal level of the ectodermal muscular layer. The faint vertical striations are epithelio-muscular cell processes. The direction of the tentacles is towards the top of the photograph. Desmoncmc nematocytes, indicated by double arrows, migrate strictly polarized with nucleus (n) forward and nematocyst capsule (c) behind with the cnidocil end trailing. A stenotele nematocyte with similar orientation is at the lower right. These nematocytes are migrating through interstitial celldepleted tissue, from a 4-hr-old implant of normal tissue located off the lower side of the photograph. The interstitial cells of the host were depleted by the following, unpublished method: Hydra were placed in a solution of 0.06% colcemid for 3 hr and then maintained 20 days in "M" solution. Under these conditions more than 99% of interstitial cells, nematoblasts and nematocytes become depleted, mainly through phagocytosis and digestion by epithelial cells, x 1,240. In longitudinal and cross-sections of the body column, migrating nematocytes are seen in rather precise longitudinal or crosssection (Figs. 6, 7). This again implicates the epithelial cell muscular process in guiding nematocytes, for there are no other structures as uniformly oriented as the nematocytes. 529 In the tentacles, nematocyte orientation is not as constant. Nematocytes are found pointing both distally and proximally and many are oriented in other directions. This suggests that nematocytes in the tentacles do considerable wandering before being incorporated into a battery. Interstitial cells: Interstitial cells have classically been considered as highly mobile cells, easily recruited to sites of wounding or budding. However, this view of the interstitial cells arose at a time when it was believed that they were the only cells active in growth and tissue repair. Kanajew's (1930) important study represents the first and still crucial demonstrations that most morphogenesis and tissue architecturing is due to epithelial cells, not to interstitial cells. This invalidates the major historical reason for supposing that interstitial cells are migratory. The available evidence for interstitial cell migrations is mainly histological and provides little information concerning the extent of cell movements. The meager ex- FIG. 5. Cell migration and displacement in Hydra littoralis. These graphs indicate the distribution of labeled cells in two hydra, one of which had been labeled with tritiated thymidine, 3 days after exchanging hydranths and distal columns. In both animals mature, migrating nematocytes are abundant in the distal column and in the tentacles (not shown) . No labeled interstitial cells were found outside regions where epithelial cells were labeled, indicating no detectable interstitial cell migration. (From Campbell, 1967c.) 530 RICHARD D. CAMPBELL perimental data, in fact, suggest that interstitial cell migration is ordinarily rather limited. Some classical histological studies dealt with interstitial cell migration by describing the appearances of these cells. These studies have been generally interpreted to be consistent with the notion that interstitial cells are amoeboid. However, it has been cautioned (Campbell, 1967c) that interstitial cell shapes, generally tear-drop or lanceolate, do not actually resemble those of most amoeboid cells. The cell shape is due to compression within the intercellular spaces. Interstitial cells do not have filopodia, and electron microscopists uniformly consider interstitial cell cytoplasm nearly devoid of organelles (Slautterback and Fawcett, 1969; Lentz, 1966) FIG. 7. Nematocyte migration in Hydra attenuata seen in longitudinal section (parallel to the axis of the hydra) . This desmoneme nematocyte is migrating towards the lower left, in the direction of the tentacles. The leading edge is occupied by a pseudopod, which is typically smaller than the one shown here. The nematocyst capsule trails with the cnidocil end (extreme upper right) pointed backwards. The cell is separated from the mcsolamella (in) by a muscular process of an epithelial cell. Ectoderm is to the upper left, endoderm to the lower right. X4,000. FIG. 6. Nematocyte migration in Hydra attenuata seen in cross section (perpendicular to the axis of the hydra) . Two desmoneme nematocytes (arrows) are migrating adjacent to each other, one (the upper one, with nematocyst capsule visible) slightly ahead of the other (lower one, with nucleus visible) . They are separated from Ihc mesolamella (m) only by epithelio-muscular cell processes, although the lower nematocyte makes a minute contact with the mesolamella. Ectoderm is to the right, endoderm to the left, x4,000. in contrast to many active cells. Some histological studies infer interstitial cell movements from changes in cell distributions under experimental or unusual situations. For example, Tardent (1954) found that during regeneration of an hydranth, interstitial cells gradually become depleted in a graded pattern along the column; this was presented as evidence that interstitial cells migrate distally during regeneration and there differentiate. Similar statements have been made about gonadogenesis, where interstitial cells become more abundant in the vicinity of presumptive gonads and become depleted elsewhere in the body. The reason why no histological study of this type provides evidence for inter- CEIX MOVEMENTS IN HYDRA stitial cell migration is that there are a variety of activities in addition to migration which can affect interstitial cell differentiation, abundance, and distribution. Interstitial cells proliferate and differentiate and their relative abundance is altered by epithelial cell proliferation. Unless one determines simultaneously the rates of these various activities, it is not possible to conclude that altered distributions of interstitial cells reflect migration. Most experimental work on interstitial cell migration has involved abnormal hydra, generally X-irradiated or nitrogen mustard-treated individuals. Results from these experiments are spectacular and unusual and will be dealt with in the next section because they do not deal with normal hydra development. Only three studies have been directed specifically at experimentally detecting interstitial cell migration in normal hydra. Both involved grafting together complementary pieces of hydra taken from unlabeled animals and from animals labeled with the nuclear isotopic marker 3H-thymidine. Tardent and Morgenthaler (1966) grafted basal, labeled hydra halves to upper, unlabeled halves and at intervals determined the total number of labeled cells in the distal region. Cell migration began within 3 hr of grafting and by 27 hr after grafting, a maximum of 137 labeled interstitial cells had advanced into the distal portions. This must have represented several hundred migrating cells because not all of the proximal cells were labeled. Tardent and Morgenthaler's experiments uphold the classic view that interstitial cells are able to migrate. Their data furthermore suggest that in a normal hydra interstitial cell migration is somewhat limited. Although Tardent and Morgenthaler did not give data on the total number of cells in these hydra, data from Bode et al. (1973) for this strain of hydra indicate that there are 10,000 to 25,000 interstitial cells and nematoblasts* in the * Tardent and Morgen thaler's term "interstitial cell" includes "large" and "small" interstitial cells and probably many nematoblasts as defined by David (1973) and Bode et al. (1973). 531 lower (labeled) half of these animals. Therefore, either only one or several per cent of the cells moved from the lower half to the upper half during Tardent and Morgenthaler's experiments, or else a substantial number of interstitial cells moved but only 10 to 20 m^. The observations do show that there are not ordinarily largescale or distant interstitial cell migrations during normal hydra development. Similar experiments were carried out for 1, 3, and 9 days by Campbell (1967c) using H. littoralis. No migratory interstitial cells were observed. According to the methods used, this finding would set migratory cells as less than 1% of the interstitial cells. Herlands (1974) furthermore showed that migration is primarily towards hypostomes. Passive Movement: Translocation of cells relative to adjacent cells or tissues propelled by activities of other cells Bulk movement of cells located in the interstitial spaces. In some circumstances, massive movements of cells take place through the intercellular spaces. The beststudied examples occur when a piece of normal hydra tissue is grafted onto a hydra whose interstitial cells have been experimentally removed. Interstitial cell depletion can be produced by X-irradiation (Zawarzin, 1929; Evlakhova, 1946; Brien and Reniers-Decoen, 1955), or by treatment with nitrogen mustard (Diehl and Burnett, 1964) or colchicine (Campbell, unpublished). Histological examination of chimeras consisting of normal and depleted tissue reveals massive interstitial cell migration into the depleted tissue (Brien and Reniers-Decoen, 1955) (Fig. 8). Migration occurs in heterografts (Diehl and Burnett, 1966) (Fig. 9) and into tissue of opposite sex (Brien, 1953) as well as in homografts. The rate of migration is impressive, although most investigators have not counted the numbers of cells which reinvade the depleted tissue. While Tardent and Morgenthaler (1966) found no difference between the number of cells migrating into X-irradiated and normal hosts, figures and descriptions in the litera- 532 RICHARD D. CAMPBELL depleted tissue is that all classes of interstitial cells and nematoblasts take part (Diehl and Burnett, 1966; Tardent and Morgenthaler, 1966) (Figs. 8, 9). These include (after the terminology of David, 1973) the big interstitial cell, which presumably includes the indeterminate stem cell; the little clustered interstitial cells, which are probably determined to become nematocytes but are not yet differentiated; and nematoblasts. While large interstitial cells sometimes have tear-dropped shapes and could perhaps be considered as amoeboid, little interstitial cells and nematoblasts have no trace of appearance of amoeboid cells and it is scarcely reasonable to suppose that they can actively migrate. FIG. 8. Interstitial cell migration from a small plug of normal tissue (between large arrows) into a host hydra whose interstitial cells had previously been eliminated by using nitrogen mustard. Thirtysix hours after implantation of the normal tissue, the animal was fixed and stained with toluidine blue. The small arrows point to numerous interstitial cells, which have migrated outwards from the implant in all directions. The two uppermost arrows are near the most distal extent of migrating cells. The limits of the implant can be seen by irregular black dots which represent carbon particles (Campbell, 1973) in the implanted epithelial cells. The large round black dots throughout the host are stenotele nematocysts. Large and small interstitial cells and nematoblasts have migrated. Both the donor and host are Hydra alienuata. x 120. ture strongly suggest that depletion of interstitial cells promotes interstitial cell movements. In fact, all investigators who have studied this problem state that this migration is so extensive that the depeleted tissue rapidly regains its normal histological aspect (see also legends of Figs. 8,9). This high rate of movement distinguishes this type of migration from that occurring in normal hydra. A second and crucial characteristic of interstitial migration into interstitial cell- FIG. 9. Migration of interstitial cells from normal Pehnatohydra oligactis tissue (below large arrows) into nitrogen mustard-treated Hydra attemiala tissue (above large arrows) . The P. oligactis tissue is marked by carbon particles in the epithelial cells, indicated by irregular dots at bottom of photograph. The large round dots throughout the photograph are stenotele nematocysts. The small arrows point to interstitial cells which have migrated from the P. oligactis tissue into the H. altenuala host during 36 hr following the grafting. x!80. CELL MOVEMENTS IN HYDRA Another characteristic of bulk movement of interstitial cells is that it proceeds very rapidly for a day or two, and then slows greatly or stops. Migration dependent upon single, locomoting cells would not be expected to show this short term behavior. The bulk movement of interstitially located cells, including the presumably non-migratory, nested nematoblasts, suggests that a distinct type of cell movement operates in these situations and that the moving cells themselves are not providing the propelling force. As yet the mechanisms of this migration are unknown. The interstitial spaces in hydra form one continuous network and bulk migration seems to occur whenever normal tissue is grafted onto tissue whose interstitial spaces are empty. It is possible that pressure or "kneading" exerted by the epithelial cells is responsible for equalizing interstitial cell distribution; grafting situations which produce a discontinuity in interstitial space packing thus seem to result in massive movement of cells. Another situation has been reported where mass movement of cells might be occurring. Moore (1971) and Moore and Campbell (1973b) studied the ability of normal tissue to induce budding in nonbudding strains of hydra. It was found that interstitial cells, and perhaps nematoblasts, move from the normal tissue into the regions of developing, induced buds in great numbers. This could be a characteristic of these unusual grafting arrangements, but it is possible that in normal hydra there is continuous bulk movement of interstitial cells from the proximal tissue into devolping buds. This could help explain the paucity of interstitial cells in the stalk. During oogenesis, large numbers of oocytes fuse to form the egg cell which is thus a huge complex mass spreading through the intercellular spaces. The fusing oocyte may extend more than half-way around the hydra column. Just before the meiotic divisions, the oocyte becomes rapidly (in a few hours) consolidated into a spherical cell. The force of this consolida- 533 tion is so strong that it ruptures the epithelial surface and the egg squeezes out to the surface of the hydra. The egg has been termed as amoeboid on the basis of this retraction and its appearance, but its amoeboid appearance is due to the spatial pattern of oocyte fusion (Brien and R.eniers-Decoen, 1950) and not due to the cell's behavior. The histological evidence is more compatible with the "retraction" being due to a squeezing by peripheral epithelial cells, forcing large oocyte mass into a central mass (Moore, unpublished). If so, egg consolidation might be related in mechanism to bulk interstitial cell migrations. Mounted nematocytes and nerves. Other types of movement which are probably completely passive are exhibited by cells which are intimately connected to epithelial cells: nerves and mounted nematocytes. These cells are presumably carried with the displacing epithelial tissues. Although these passive cell movements have not been observed directly, their existence is in accord with cell production and turnover kinetics. Germinal cells in tesles. In testes, there is a graded germinal differentiation sequence with proliferating spermatogonia near the mesolamella surface and mature spermatozoids near the apical surface (Brien and Reniers-Decoen, 1951; Schincariol and Habowsky, 1972). Thus, during periods of spermatogenesis there is continual displacement of differentiating cells in an apical direction. While this cell movement is not long in range compared to other migrations in hydra, it is exceptionally well ordered and probably critical to tissue functioning. Cellular movements towards gonads during early stages of the sexual phase. It is commonly stated that interstitial cells, or their derivatives, migrate to sites of gonad formation, thus resulting in the local swelling of the gonads. However, this migration has never been experimentally documented and from kinetic and histological considerations might even be questioned. The extensive and long mitotic activity of gonial cells preceding testis for- 534 RICHARD D. CAMPBELL mation (Schincariol and Habowsky, 1972) indicates that the primary source of these cells is local proliferation. In some species (e.g., Hydra pirardi and Pelmatohydra oligactis) testes cover the entire body column, thus ruling out recruitment of germinal cells from adjacent regions. In ovaries, cell accumulation is due to final consolidation of the egg cell (see above) rather than interstitial cell migration. Thus, although cell accumulation in gonads has been long described as due to cell migration, there has never been experimental evidence for considering that it is so. CONCLUSIONS All cells in a hydra are moving, in complete and continual tissue renewal patterns, with the various cell types showing different, and often several simultaneous, locomotory patterns. The mechanisms range from active to passive and are intimately tied in with the developmental pattern of the animal. Advances in cell recognition, marking, and manipulation appear now to enable us to determine definitely and describe all the cell locomotory pathways in hydra. Recent experimental work indicates that the classical histological analyses and concepts regarding cell movements in hydra were at times misleading. Thus, the early considerations that epithelial cells represented a fixed framework of the adult hydra were completely reversed by Brien's demonstration of continuous epithelial cell turnover. 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