Interstitial cell (I-cell) migration in hydra is essential for establishment of the regional cell differentiation pattern in the organism. All previous in vivo studies have indicated that cell migration in hydra is a result of cell-cell interactions and chemotaxic gradients. Recently, in vitro cell adhesion studies indicated that isolated nematocytes could bind to substrata coated with isolated hydra mesoglea, fibronectin and type IV collagen. Under these conditions, nematocytes could be observed to migrate on some of these extracellular matrix components. By modifying previously described hydra grafting techniques, two procedures were developed to test specifically the role of extracellular matrix components during in vivo I-cell migration in hydra. In one approach, the extracellular matrix structure of the apical half of the hydra graft was perturbed using β-aminopropi-onitrile and β-xyloside. In the second approach, grafts were treated with fibronectin, RGDS synthetic peptide and antibody to fibronectin after grafting was performed. In both cases, I-cell migration from the basal half to the apical half of the grafts was quantitatively analyzed. Statistical analysis indicated that β-aminopropionitrile, fibronectin, RGDS synthetic peptide and antibody to fibronectin all were inhibitory to I-cell migration as compared to their respective controls.

β-xyloside treatment had no effect on interstitial cell migration. These results indicate the potential importance of cell-extracellular matrix interactions during in vivo I-cell migration in hydra.

Extracellular matrix (ECM) plays important roles in development not only by providing structural support to cells but also by influencing such cellular processes as cell proliferation, cell differentiation, cell adhesion and cell migration (see review by Hay, 1991). In order to study the influences of ECM on cell migration under in vivo conditions, we have utilized the fresh water invertebrate, Hydra vulgaris as a model system for analysis. Hydra was chosen because of its simple body structure and its high capacity for regeneration. Structurally, hydra is composed of a gastric tube with a head at one pole and a foot process at the other pole. Its entire body wall is formed by an epithelial bilayer with an intervening ECM termed the mesoglea (Campbell and Bode, 1983). Hydra mesoglea is known to contain the major ECM components (i.e. fibronectin, laminin, type IV collagen and heparan sulfate proteoglycan) found in vertebrate and more complex invertebrate species (Sarras et al., 1991a). Recent functional studies have demonstrated that these ECM components play an important role in hydra head regeneration (Sarras et al., 1991b) and in hydra cell aggregate development (Sarras et al., 1993).

The regional cell differentiation pattern of hydra is dependent on migration of interstitial cells (I-cells). Under in vitro conditions, Day and Lenhoff (1981) have demonstrated that hydra cells attach to and spread on isolated mesoglea. More recently, Agosti and Stidwill (1991) have demonstrated that hydra nematocytes attach to and migrate on substrata coated with isolated mesoglea or coated with purified ECM components such as type IV collagen and laminin. Hydra nematocytes have also been shown to bind to fibronectin in a RGD-dependent manner (Ziegler and Stidwill, 1992). Previous in vivo studies by Campbell and Marcum (1980) also indicated that nematocytes migrate between ectodermal epitheliomuscular cells via cell-cell contact guidance mechanisms. While all previous in vivo studies have indicated that cell migration in hydra depends on cell-cell interactions (Campbell and Marcum, 1980) and chemotaxic gradients (Teragawa and Bode, 1991), we propose that cell-ECM interactions are also critical to this process.

In order to determine if hydra cell-ECM interactions do occur in situ, we have developed an in vivo bioassay that combines a number of previously published procedures. One procedure involves the use of hydra grafting techniques that allow quantification of the migration of I-cells from a donor hydra (basal half of graft) to a host hydra (apical half of graft) (Teragawa and Bode, 1990, 1991). This technique was then combined with two other procedures. In one case, hydra grafts were made using animals in which the structural integrity of the mesoglea was perturbed using drugs that interfere with the synthesis and processing of matrix components. In the second case, procedures were employed that allow one to introduce macromolecules between the epithelium and mesoglea utilizing a dimethylsulfoxide (DMSO)-loading procedure (Fraser et al., 1987). Passage of macromolecules between epithelial cells in hydra is normally prevented by septate junctions (Wood and Kuda, 1980), but low levels of DMSO have been shown to temporarily open these junctions (Hans Bode, personal communication). We utilized this DMSO loading procedure to introduce macromolecules into hydra grafts that could potentially interfere with normal cell-ECM interactions (e.g. ECM components, synthetic peptide, or anti-bodies to ECM components). Using these approaches, the current study therefore focused on the effect of alterations in ECM structure and the role of fibronectin on in vivo I-cell migration in hydra.

Culture of animals

Hydra vulgaris (previously named Hydra attenuata) were used in all experiments. Animals were cultured in hydra medium (HM) as pre-viously described by Sarras et al. (1991a).

Depletion of I-cells in hydra using hydroxyurea and use of drugs to alter mesoglea structure

To deplete hydra of I-cells, similar size hydra polyps (about 30 per group) were incubated in HM containing 0.01 M hydroxyurea (HU; Sigma Chemical Company, St Louis, MO) for 5 days with repeated solution changes. Animals were then transferred into fresh HM (without HU) for 2 days to allow recovery of the polyps from HU treatment prior to use. Under these conditions, the I-cell population is reduced by approximately 99% as monitored by macerate analysis and as reported by Bode et al. (1976) and Teragawa and Bode (1990). I-cell-depleted hydra were used as host grafts (apical half of graft) in the cell migration assay.

To disrupt mesoglea structure, two protocols were followed. In the first, polyps were treated with either 0.05 mM β-aminopropionitrile (β-APN) (Sigma) or 0.01 mM p-nitrophenyl β-D-xylopyranoside (β-xyloside; Sigma) for 15 days. 0.01 M HU was added to the above solutions at day 11. A 2-day recovery period in HM was employed after 15 days of drug and HU treatment before grafting was performed. In the second protocol, although the length of treatment time was the same as previously described, the sequence of drug treatment was reversed (i.e. polyps were first treated with HU and then with β-APN or β-xyloside) before grafting was performed. Previous biochemical and autoradiography studies have shown that mesoglea structure is altered under these conditions of β-APN and β-xyloside treatment (Sarras et al., 1991b). Two different sequences of HU and β-APN or β-xyloside treatment were followed because of the concern that initial treatment with the mesoglea perturbing drugs would completely inhibit cell migration, thereby preventing the depletion of I-cells during HU treatment. As will be discussed in the results section, this was not the case.

Labeling of I-cells with 5-bromo-2′-deoxyuridine

The labeling procedure was carried out according to Teragawa and Bode (1990, 1991). Briefly, animals were injected through the hypostome into the gastric cavity with an aqueous solution containing 1.0 mM 5-bromo-2′-deoxyuridine (BrdU; Sigma) and 1–2% India ink (Pelikan C11/1431a, Bio/medical Specialties). As an analog of thymidine, BrdU is incorporated into DNA during S-phase. BrdU-labeled cells can be distinguished from non-BrdU cells with immuno-histochemical staining using antibody against BrdU. In hydra, both ectodermal and endodermal cells can be labeled after 12 hours following BrdU injection (Teragawa and Bode, 1990). India ink can be phagocytosed by endodermal epithelial cells and, thus, was used in our grafting experiments to distinguish the graft junction line between BrdU-labeled tissues and those non-BrdU labeled. In order to reduce experimental variations, efforts were taken to select similar size polyps without buds for each experiment. Buds were removed before grafting from any animal that entered the budding process after being selected and during pregrafting treatment. Considering the S-phase in hydra interstitial cells is 12 hours (Campbell and David, 1974), BrdU injections were carried out 12 hours before grafting and repeated 1 hour before grafting began. These BrdU-injected animals were used as I-cell donors (basal half of graft) in hydra grafts.

Grafting procedures

The grafting procedure is illustrated in Fig. 1. For these experiments, the division between the apical and the basal halves is determined by the boundary between the gastric region and the budding region as described by Campbell and Bode (1983). The grafting techniques that we employed were based on the procedures described by Teragawa and Bode (1990, 1991) with the following modifications: (1) the basal half of a BrdU/ink-labeled hydra polyp was always grafted to the apical half of a non-BrdU/ink-labeled polyp that had been either HU or HU and drug treated; (2) for any animals that grew buds during the pregrafting treatment, all buds were removed before animals were grafted; and (3) grafted tissues were held together on a fishing line with two end pieces of parafilm for 2 hours before any further treatment was performed.

Fig. 1.

Illustration of the in vivo I-cell migration assay which involves hydra grafting techniques. Group A hydra were treated with 0.01 M hydroxyurea (HU) for 5 days to eliminate their I-cell population. A subgroup of hydra polyps were double treated with 0.05 mM β-APN or 0.01 mM β-xyloside for 15 days to perturb their mesoglea structure and then with HU for 5 days. A second subgroup of hydra were treated with this order reversed (i.e. 5 days HU treatment followed by 15 days of β-APN or β-xyloside treatment). Group B hydra were labeled with 1 mM BrdU containing 1% India ink at 12 hours and 1 hour prior to grafting. The basal half of BrdU-labeled polyps was always grafted with the apical half of HU-treated or double-treated polyps and the grafts were kept in hydra medium for 24 hours. The determination of the cutting line separating apical and basal grafts is described in ‘Materials and Methods’. In a separate set of experiments, non-β-APN- or non-β-xyloside-treated hydra grafts were treated on ice for 30 minutes with reagents (fibronectin, synthetic peptides or antibodies) containing 5% DMSO at 2 hours after grafting. After washes in DMSO-free reagent solutions, hydra grafts were incubated a total of 24 hours in each reagent solution. All grafts were processed for immunocytochemical staining using BrdU antibody and viewed with a light microscope containing a camera lucida attachment.

Fig. 1.

Illustration of the in vivo I-cell migration assay which involves hydra grafting techniques. Group A hydra were treated with 0.01 M hydroxyurea (HU) for 5 days to eliminate their I-cell population. A subgroup of hydra polyps were double treated with 0.05 mM β-APN or 0.01 mM β-xyloside for 15 days to perturb their mesoglea structure and then with HU for 5 days. A second subgroup of hydra were treated with this order reversed (i.e. 5 days HU treatment followed by 15 days of β-APN or β-xyloside treatment). Group B hydra were labeled with 1 mM BrdU containing 1% India ink at 12 hours and 1 hour prior to grafting. The basal half of BrdU-labeled polyps was always grafted with the apical half of HU-treated or double-treated polyps and the grafts were kept in hydra medium for 24 hours. The determination of the cutting line separating apical and basal grafts is described in ‘Materials and Methods’. In a separate set of experiments, non-β-APN- or non-β-xyloside-treated hydra grafts were treated on ice for 30 minutes with reagents (fibronectin, synthetic peptides or antibodies) containing 5% DMSO at 2 hours after grafting. After washes in DMSO-free reagent solutions, hydra grafts were incubated a total of 24 hours in each reagent solution. All grafts were processed for immunocytochemical staining using BrdU antibody and viewed with a light microscope containing a camera lucida attachment.

Treatment of hydra grafts with reagents

Hydra grafts were removed from the fishing lines 2 hours after grafting and transferred into microtiter plates (Nunc, Denmark) with 1 graft per well. The microtiter plates were chilled to 4°C by placing them on an ice water bath. The hydra medium in the wells were removed by aspiration with a tuberculin syringe and reagent solutions containing 5% DMSO were added into each well with 10 μl/well. DMSO was used to introduce reagents between the epithelium and the mesoglea because it is able to open septate junctions between hydra epithelial cells (Fraser et al., 1987; Hans Bode, personal communication). The following reagents were tested in grafting treatments: bovine serum albumin (BSA; Sigma) at 0.05 mg/ml, fibronectin (Collaborative research Inc. Bedford, MS) at 0.05 mg/ml, GRGDSP (Gly, Arg, Gly, Asp, Ser, Pro) and GRGESP (Gly, Arg, Gly, Glu, Ser, Pro) synthetic peptides (Telios, San Diego, CA) at 0.5 mg/ml, and non-immune rabbit serum or polyclonal antibody against human plasma fibronectin (ICN Biomedicals) at 1:10 dilution. DMSO loading of reagents was carried out on ice for 30 minutes before the solutions were removed and fresh non-DMSO-containing reagent solutions were added to the wells. This latter step was needed (1) to prevent any leakage of the loaded reagents from the inter-epithelial space before septate junctions closed and (2) to dilute the residual DMSO in hydra body. Hydra grafts remained on ice for additional 15 minutes after DMSO treatment to allow septate junction closure and recovery. The non-DMSO reagent solutions were changed one more time to further dilute residual DMSO and hydra grafts were left in the final change of non-DMSO reagent solutions at 18 °C until 24 hours had elapsed from the initial time of reagent loading.

Immunocytochemistry and quantitative analysis of in vivo I-cell migration

At 24 hours after grafting and reagent treatment, hydra grafts were processed for immunohistochemistry staining following the procedures described by Teragawa and Bode (1990). Specimens were examined with a bright-field light microscope (Nikon) fitted with a camera lucida attachment. The junction line between the apical and basal half of the grafts was recognized by the ink labeling of the basal graft. Migration was considered if a BrdU-positive cell appeared above the junction line in the apical graft. Because the body wall of the hydra body column is composed of an epithelial bilayer, two epithelial bilayers are compressed together in whole-mount preparations. By focusing through the two epithelial bilayers in hydra whole-mount preparations, all BrdU-positive I-cells in the apical graft were individually identified and traced using the camera lucida drawing attachment. Cells in these camera lucida drawings were then counted for quantitative analysis. In order to avoid experimental error created by any ambiguity in the ink line, the apical half was divided into ten equal regions. Any BrdU-positive cells that appeared in the region next to the ink line in the apical half were not counted as migrated cells. A minimum of three grafts was used per parameter in each experiment and all experiments were repeated at least three times. The total number of BrdU-positive cells within each apical graft was counted and these numbers were normalized with each experiment as percent of control. Only the percent of control was used when data from different experiments was combined for statistical analysis. Based on the combined percent of control, either a student t-test or an ANOVA test was used to compare the control and experimental groups depending on the number of groups analyzed. A P value ≤0.05 was set as the level of significant difference.

Ultrastructural analysis of DMSO-loaded hydra

To examine the effect of DMSO loading on the attachment of epithe-lial cells to the extracellular matrix, scanning (SEM) and transmission (TEM) electron microscopy was performed. Hydra were DMSO loaded with BSA or test molecules such as fibronectin and then processed for SEM or TEM at various time points (0–24 hours after treatment) as previously described by Sarras et al. (1993). For SEM analysis, hydra were freeze-fractured (Sarras et al., 1993) to allow the epithelial bilayer to be observed in either a transverse or coronal plane.

Perturbation of mesoglea collagen cross-linking by β-APN inhibited I-cell migration

The hydra grafting method utilized in this study is illustrated in Fig. 1. With this method, BrdU-positive cells appearing in the host hydra (apical half of the grafts) are solely due to migration of cells from the donor hydra (basal half of the graft). Illustrations of these grafts and the appearance of migrated cells in the apical half of the grafts is shown in Figs. 2 and 3. The total number of BrdU-positive cells within the apical half was used for quantitative analysis of cell migration. Under the BrdU-labeling conditions used, the present study focused on I-cell migration. As shown in Fig. 4, I-cell migration was significantly reduced in host (apical) grafts treated with β-APN. Because β-APN treatment reduced but did not totally inhibit I-cell migration, the order in which hydra were treated with HU and β-APN did not significantly alter the final results. In contrast, no significant differences were observed between control and β-xyloside-treated groups. These results indicated the relative importance of collagen cross linking versus proteoglycan processing during I-cell migration in hydra.

Fig. 2.

Whole-mount preparations of control (A,C,D) and experimental (B) grafts stained with antibody to BrdU are shown. An experimental graft DMSO-loaded with fibronectin (0.05 mg/ml) is shown in B while the control shown represents a grafted DMSO-loaded with BSA at the same concentration. At low magnification, the juncture point between the apical (the host half for cell migration) and basal (donor half which was injected with BrdU and ink-labelled) halves of the graft are indicated by the arrows (shown in A and B). BrdU-labelled nuclei of I-cells, which had migrated from the basal to the apical half, are indicated by the arrowheads in A and B. At higher magnification, BrdU-labelled I-cell nuclei in the apical half could be distinguished from one another (C). These I-cells reside in the epithelial bilayer of the tubular body column of hydra. By focusing through the two epithelial bilayers, which are compressed together in whole-mount preparation, I-cells can be identified and the total number that had migrated into the apical half of the graft counted. As viewed at the periphery of the graft using phase-contrast microscopy (D), some I-cells (arrowheads) are seen in juxtaposition to the extracellular matrix (arrows), which appears as a clear line at the base of the ectodermal cell layer in these preparations. Bar for A and B, 125 μm. Bar for C and D, 25 μm.

Fig. 2.

Whole-mount preparations of control (A,C,D) and experimental (B) grafts stained with antibody to BrdU are shown. An experimental graft DMSO-loaded with fibronectin (0.05 mg/ml) is shown in B while the control shown represents a grafted DMSO-loaded with BSA at the same concentration. At low magnification, the juncture point between the apical (the host half for cell migration) and basal (donor half which was injected with BrdU and ink-labelled) halves of the graft are indicated by the arrows (shown in A and B). BrdU-labelled nuclei of I-cells, which had migrated from the basal to the apical half, are indicated by the arrowheads in A and B. At higher magnification, BrdU-labelled I-cell nuclei in the apical half could be distinguished from one another (C). These I-cells reside in the epithelial bilayer of the tubular body column of hydra. By focusing through the two epithelial bilayers, which are compressed together in whole-mount preparation, I-cells can be identified and the total number that had migrated into the apical half of the graft counted. As viewed at the periphery of the graft using phase-contrast microscopy (D), some I-cells (arrowheads) are seen in juxtaposition to the extracellular matrix (arrows), which appears as a clear line at the base of the ectodermal cell layer in these preparations. Bar for A and B, 125 μm. Bar for C and D, 25 μm.

Fig. 3.

Two sample camera lucida drawings of a control graft (A) and a graft treated with β-APN (B). For quantitative analysis of I-cell migration, the apical half and the juncture point with the basal half was drawn for each graft. The junction line between the two graft halves could be identified under the microscope by the ink particle labeling of the basal half. This juncture point is indicated by the arrowheads in the camera lucida drawings shown in this figure. The asterisk (*) indicates tentacles of the apical half of the graft. Bar, 180 μm.

Fig. 3.

Two sample camera lucida drawings of a control graft (A) and a graft treated with β-APN (B). For quantitative analysis of I-cell migration, the apical half and the juncture point with the basal half was drawn for each graft. The junction line between the two graft halves could be identified under the microscope by the ink particle labeling of the basal half. This juncture point is indicated by the arrowheads in the camera lucida drawings shown in this figure. The asterisk (*) indicates tentacles of the apical half of the graft. Bar, 180 μm.

Fig. 4.

The effect of drugs that perturb ECM structure on in vivo I-cell migration. Hydra polyps used for apical grafts were treated with either 0.05 mM β-APN or 0.01 mM β-xyloside for 15 days and with 0.01 M HU for 5 days (HU+β-APN or HU+β-XYL). For details, see ‘Materials and Methods’. Asterisks indicate groups statistically different from controls with P≤0.05.

Fig. 4.

The effect of drugs that perturb ECM structure on in vivo I-cell migration. Hydra polyps used for apical grafts were treated with either 0.05 mM β-APN or 0.01 mM β-xyloside for 15 days and with 0.01 M HU for 5 days (HU+β-APN or HU+β-XYL). For details, see ‘Materials and Methods’. Asterisks indicate groups statistically different from controls with P≤0.05.

In addition to the total number of migrated cells, the distance of cell migration was also analyzed. Apical grafts were divided into four zones longitudinally and BrdU-positive cells within each zone were counted and plotted. To determine if the absolute distance of cell migration was affected, migrated cells within each specific zone were compared between control and experimental groups. The results indicated that, although the total number of migrated cells was reduced in the β-APN-treated apical half, cells were still able to migrate to all four regions (most proximal to most distal). Therefore, β-APN treatment did not reduce the maximal distance cells could be seen to migrate, but it did reduce the number of cells that could effectively migrate.

Fibronectin, antibody to fibronectin and RGDS synthetic peptide inhibited I-cell migration in hydra grafts

Brief exposure to DMSO has been shown to open the septate junctions temporarily between hydra epithelial cells (Fraser et al., 1987; Hans Bode, personal communication). This procedure was used in the present study to introduce macro-molecules between epithelial cells so that their effect on in vivo I-cell migration could be analyzed. As shown in Fig. 5, I-cell migration was inhibited in hydra grafts treated with fibronectin, RGD peptide and antibody to fibronectin as compared to their respective controls (BSA, RGE peptide and non-immune serum).

Fig. 5.

The effect of DMSO-loading with fibronectin (FN), synthetic peptides (RGD or RGE) and antibody to fibronectin (Anti-FN) on I-cell migration. (A) FN and RGD peptide significantly reduced I-cell migration as compared to BSA or RGE controls. (B) Anti-FN also significantly inhibited I-cell migration as compared to non-immune serum (Non-Immune) controls. I-cell migration was analyzed as described in ‘Materials and Methods’ and in the figure legend of Fig. 3. Asterisks indicate groups significantly different from controls (P≤0.05).

Fig. 5.

The effect of DMSO-loading with fibronectin (FN), synthetic peptides (RGD or RGE) and antibody to fibronectin (Anti-FN) on I-cell migration. (A) FN and RGD peptide significantly reduced I-cell migration as compared to BSA or RGE controls. (B) Anti-FN also significantly inhibited I-cell migration as compared to non-immune serum (Non-Immune) controls. I-cell migration was analyzed as described in ‘Materials and Methods’ and in the figure legend of Fig. 3. Asterisks indicate groups significantly different from controls (P≤0.05).

Attachment of epithelial cells to the extracellular matrix was not disrupted with the drugs or reagents used in these studies

To determine if the inhibition of I-cell migration could be a secondary effect resulting from a disruption of attachment of epithelial cells to the mesoglea, ultrastructural studies were performed. It was previously shown that treatment of hydra with β-APN or β-xyloside altered the structure of hydra extracellular matrix (mesoglea) but did not disrupt the attachment of epithelial cells to the mesoglea. This analysis was expanded in the current study to evaluate the effect of the DMSO-loading procedure on the attachment of epithelial cells to the mesoglea. As shown in Fig. 6, at the concentrations used in these studies, DMSO loading of molecules such as fibronectin (0.05 mg/ml) did not cause a disruption of the attachment of epithelial cells to the extracellular matrix as evaluated by SEM or TEM analysis.

Fig. 6.

Ultrastructural analysis of the effect of the DMSO-loading procedure on the attachment of epithelial cells to the extracellular matrix in hydra. Control (A,C,E; BSA, 0.05 mg/ml) and experimental (B,D,F; fibronectin, 0.05 mg/ml) specimens are shown. As shown by SEM at low (A,B) and intermediate (C,D) magnification, neither BSA or fibronectin at the concentrations used in this study resulted in any apparent disruption of the attachment of ectodermal (Ec) or endodermal epithelial cells to the hydra extracellular matrix (arrowheads indicate the attachment sites of epithelial cells to the hydra mesoglea). The attachment of epithelial cells to the extracellular matrix was confirmed by TEM analysis of BSA-(E) and fibronectin-(F) treated specimens. The close association of the epithelial plasma membrane to the mesoglea (M) is indicated by the arrowheads in E and F. Bar for A and B, 10 μm. Bar for C and D, 2.5 μm. Bar for E and F, 233 nm.

Fig. 6.

Ultrastructural analysis of the effect of the DMSO-loading procedure on the attachment of epithelial cells to the extracellular matrix in hydra. Control (A,C,E; BSA, 0.05 mg/ml) and experimental (B,D,F; fibronectin, 0.05 mg/ml) specimens are shown. As shown by SEM at low (A,B) and intermediate (C,D) magnification, neither BSA or fibronectin at the concentrations used in this study resulted in any apparent disruption of the attachment of ectodermal (Ec) or endodermal epithelial cells to the hydra extracellular matrix (arrowheads indicate the attachment sites of epithelial cells to the hydra mesoglea). The attachment of epithelial cells to the extracellular matrix was confirmed by TEM analysis of BSA-(E) and fibronectin-(F) treated specimens. The close association of the epithelial plasma membrane to the mesoglea (M) is indicated by the arrowheads in E and F. Bar for A and B, 10 μm. Bar for C and D, 2.5 μm. Bar for E and F, 233 nm.

Among all hydra cell types, interstitial cells are the fastest dividing subgroup of cells with a S-phase of about 12 hours (Campbell and David, 1974). This feature gave us the opportunity specifically to analyze I-cell migration within hydra grafts. Previous studies have shown that treatment of hydra with HU for 5 days depletes the majority of their I-cell population (Bode et al., 1976) and, therefore, when the apical halves of these hydra are grafted to the basal halves of normal hydra, an I-cell population gradient is created. This gradient results in a higher number of cells migrating from basal half to the apical half as compared to hydra grafts formed between non-HU-treated polyps (Teragawa and Bode, 1990). The combination of this HU-induced cell migration pattern and the BrdU labeling of I-cells granted us an unique model system to determine if perturbation of ECM structure could affect I-cell migration. It should be noted that while other cell types do migrate in hydra (e.g. nemato-cytes), the current study only focused on I-cell migration because of the techniques employed. In regard to our pharmacological studies, β-APN has been shown to interfere with collagen cross linking by inhibiting lysyl oxidase (Page and Benditt, 1972; Wilmarth and Froines, 1992). Previous studies have established that β-APN does affect mesoglea structure (Sarras et al., 1993) and collagen cross linking in hydra (Sarras et al., 1991b). In addition, previous in vitro studies have shown that, when used as a substratum, collagens (especially type IV collagen) promote migration of various cell types such as bronchial epithelial cells (Rickard et al., 1993), neural crest cells (Perris et al., 1991) and hydra nematocytes (Agosti and Stidwill, 1990). Under in vivo conditions, type I, III and IV collagen were shown to appear along the neural crest migratory pathways during development of chick embryos (Perris et al., 1991). This indicated a role for these ECM components in mediating neural crest cell migration during embryonic development. Therefore, in the current study, the reduction of I-cell migration in β-APN-treated grafts can be interpreted as the result of alterations in mesoglea structure related to the per-turbation of collagen processing. β-xyloside has been shown to interfere with the addition of glucosylaminoglycan (GAG) chains to proteoglycan core proteins (Lelongt et al., 1988). When added to culture medium, this reagent inhibited the migration of primary mesenchyme cells in sea urchin embryos (Lane and Solursh, 1988). While β-xyloside treatment does inhibit hydra head regeneration (Sarras et al., 1991b) and mor-phogenesis of hydra cell aggregates (Sarras et al., 1993), its treatment had no affect on I-cell migration in hydra grafts. We can therefore eliminate alterations in cell migratory patterns as the basis for β-xyloside’s inhibitory effect on general mor-phogenesis in hydra. Overall, our results indicate that, in hydra, I-cell migration is more sensitive to alterations in collagen structure than alterations in proteoglycan structure.

Under in vitro conditions, fibronectin has been shown to promote cell adhesion and cell migration in neural crest cells (Perris et al., 1989; Dufour et al., 1988), keratinocytes (Sarret et al., 1992) and smooth muscle cells (Naito et al., 1992). Hydra nematocytes have been shown to bind to a fibronectin-coated substratum and this binding is known to be RGD dependent (Ziegler and Stidwill, 1991). Although nematocytes can bind to fibronectin, studies by Agosti and Stidwill (1990) have shown that mature nematocytes migrate poorly on this ECM component. The current study revealed that under in vivo conditions, I-cell migration can be inhibited by macromolecules that can compete with cell-fibronectin interactions (e.g. intact fibronectin, RGD peptide or antibody to fibronectin).

The inhibitory effect of fibronectin and antibody to fibronectin on I-cell migration may seem contradictory. A number of interpretations can be proposed for this result. For example, these results could result from a competition between endogenous mesoglea fibronectin and exogenously added soluble fibronectin. In this case, it can be proposed that normal I-cell migration is dependent on the interaction of cell surface fibronectin receptors with fibronectin molecules that are insoluble and bound within the three-dimensional structure of the hydra ECM. The presence of hydra cell surface receptors for fibronectin is supported by in vitro cell adhesion studies by Ziegler and Stidwill (1992). Exogenously applied soluble fibronectin would compete for I-cell ECM receptors and interfere with normal contact guidance mechanism and therefore result in an inhibition of cell migration. In contrast, antibody to fibronectin could mask endogenously bound fibronectin and thereby interfere with the ability of I-cell ECM receptors to bind to fibronectin in the ECM. These proposals are supported by the fact that RGD peptide could also inhibit I-cell migration. The RGD amino acid sequence is known to be a cell binding domain for fibronectin and can bind to integrin receptors (see review by Akiyama et al., 1990; Hynes, 1992). When studied in an in vitro assay with isolated hydra nematocytes, this peptide was shown to inhibit nematocyte binding to fibronectin (Ziegler and Stidwill, 1992). These observations can now be extended to the in vivo situation in the case of migrating I-cells. The inhibitory affect of RGD peptide on I-cell migration appeared to be specific since the inactive peptide RGE had no effect on I-cell migration.

Although the total number of migrating cells is reduced after treatment with β-APN, fibronectin, anti-fibronectin antibody or RGDS synthetic peptides, the maximal distance of I-cell migration, however, was not affected. One explanation for this result is that the migration of particular subpopulations of I-cells was significantly inhibited by these reagents while other subpopulations of I-cells were only marginally affected or were not affected at all. While the immunocytochemical procedures utilized in this study present I-cells as a morphologically homogeneous group, they are in fact a heterogeneous cell population composed of multipotent stem cells and various cell lineage precursor cells (Heimfeld and Bode, 1986a,b). These subpopulations of cells could be selectively sensitive to the perturbing reagents used due to the expression of specific cell surface receptors for ECM components.

Several mechanisms have been proposed to explain cell migration in hydra. These mechanisms include (1) the mechanical forces resulting from tissue changes during contraction and expansion of polyps (Teragawa and Bode, 1990), (2) cell-cell interactions involved in contact guidance (Campbell and Marcum, 1980) and (3) external cues such as chemotactic signaling that may be related to the head activator gradient along the longitudinal axis of the organism (Teragawa and Bode, 1991). In addition to these proposed mechanisms, our data indicate the potential role cell-ECM interactions during in vivo I-cell migration. The inhibition of I-cell migration observed in this study could reflect a direct interaction of I-cells with the ECM or could result from alterations in the normal attachment of epithelial cells with the ECM, which then causes a secondary inhibitory effect on I-cell migration. In the latter case, altered epithelial attachment to the ECM would result in a perturbation of cell-cell interactions and/or chemo-tactic signaling systems that normally occur during I-cell migration. The ultrastructural analyses performed in this study however, indicate that, under the conditions used, no disruption in the attachment of epithelial cells with the mesoglea could be observed. While this in itself does not exclude potential secondary inhibitory effects, taken in concert, all of the data presented in the current study is consistent with a direct interaction of I-cells with ECM components. As a final note regarding epithelial-ECM interactions in hydra, it should be noted that DMSO loading of higher concentrations of fibronectin (e.g. 0.1 mg/ml) can cause a rapid dissociation of hydra cells in the adult organism (data not shown). This suggests that, while epithelial-fibronectin interactions may be a component of epithelial-ECM attachment in hydra, these interactions were not affected by the concentration of reagents used in the present study. Although the exact mechanisms underlying in vivo I-cell migration in hydra are not yet clear, the current study and others do point to the presence of specific ECM cell surface receptors within the different hydra cell types. In this regard, Ziegler and Stidwill (1992) have isolated integrin-like plasma membrane proteins from nematocytes with binding affinity for fibronectin. Further studies will be required to identify the full spectrum of ECM cell surface binding proteins among the different hydra cell types and to determine their respective roles in the process of pattern formation in this organism.

The authors wish to thank Dr Hans R. Bode for his continuous support and Drs Lynne Littlefield and Hiroshi Shimizi for their help and suggestions regarding hydra grafting and quantitative analysis of in vivo I-cell migration. The authors also wish to thank the technical support of Jacquelyn K. Huff in regard to the ultrastructural studies presented in this article. The studies described in this article were supported by funds provided by NIH (RR06500) and the International Juvenile Diabetes Foundation Inc.

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