Epithelial interactions in Hydra: apoptosis in interspecies grafts is induced by detachment from the extracellular matrix
Zoologisches Institut, Christian-Albrechts Universität zu Kiel, 24116 Kiel, Germany
Author for correspondence (e-mail: tbosch{at}zoologie.uni-kiel.de)
Accepted 16 September 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Hydra, histocompatibility, allorecognition, apoptosis, anoikis, extracellular matrix, septate junction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the wild, some invertebrates undergo so called `natural
transplantations', when they experience close contact with tissue from other
individuals of the same or related species during larval settlement and growth
(Rinkevich, 1996). Due to the
presence of mobile multipotent stem cells in these animals, tissue fusion may
lead to formation of chimeric individuals. To prevent such somatic and germ
cell parasitism, efficient detection and defence mechanisms against non-self
cells are thought to have evolved (Buss,
1982
). Cnidaria, as the most basal eumetazoans, have no
specialised immune cells. However, as shown in the hydroid Hydractinia
echinata, contact between two colonies leads to either fusion or
rejection. Since Hydractinia fuse only with self and reject all
non-related conspecific tissue (Frank et
al., 2001
), they seem to possess a genetic system that can
discriminate between self and non-self
(Mokady and Buss, 1996
). The
solitary growing freshwater polyp Hydra also was suggested to have
such a system (Bosch and David,
1986
) and grafts between different Hydra species have
long been considered a model for the ancestral form of transplantation
immunity (Kolenkine, 1958
;
Kanaev, 1969
;
Campbell and Bibb, 1970
). In
heterografts between H. attenuata (=H. vulgaris) and H.
oligactis we observed an increased number of phagocytozing epithelial
cells, located within the contact zone, that selectively eliminate cells from
the other species (Bosch and David,
1986
). This observation suggested that a fundamental aspect of
immunity the ability to distinguish self from non-self is
present in Hydra. This was surprising since promiscuous fusion
between individual Hydra polyps never occurs naturally and,
therefore, the risk of cell lineage parasitisms is very low in
Hydra.
To resolve the issue of the presence of a discriminative allorecognition system in Hydra, we reinvestigated tissue interactions between Hydra vulgaris and Hydra oligactis. We observed that a large number of apoptotic cells accumulate in the contact region of interspecies grafts. Initiation of apoptosis at the graft site is correlated with impaired cellmatrix and cellcell contacts. We report elsewhere (S. G. Kuzetsov and T. C. G. Bosch, manuscript in preparation) that contact to allogeneic tissue does not evoke any response in Hydra in terms of phagocytosis and elimination on non-self cells. We therefore suggest that, contrary to the previous view, in interspecies grafts apoptosis is induced by impaired cellcell or cellmatrix contacts and not by a discriminative recognition system.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transplantation procedure
Grafts were produced as described
(Fujisawa et al., 1990).
Briefly, animals were cut transversely in the mid-gastric region, and
complementary apical and basal halves of two polyps were threaded on a fishing
line and held together by sleeves of polyethylene tubing to facilitate
healing. After 1-2 h grafts were taken off the fishing line and maintained
under standard conditions without feeding.
Preparation of mixed-cell aggregates
Polyps were mechanically dissociated into a cell suspension in dissociation
medium and reaggregated as described
(Gierer et al., 1972). To
prepare heteroaggregates, about 250 polyps of Hydra vulgaris and
Hydra oligactis were separately dissociated in 20 ml dissociation
medium. Tissue pieces and large cell clusters were allowed to sink for 30s.
Polypropylene microcentrifuge tubes (Roth) were filled with 400 µl of the
remaining cell suspension and centrifuged at 1200 g for 5 min
at 18°C. The size of the resulting pellets allowed us to assess and
equilibrate cell concentrations in both preparations. For heteroaggregates,
equal volumes of Hydra vulgaris and Hydra oligactis cell
suspensions were carefully mixed together, transferred to microcentrifuge
tubes and pelleted as described above. The dissociation medium was then
diluted with the hydra medium to 75%, 50%, 33%, 25%, 10%, 5% in 6, 8, 20, 24,
32 and 36 h after reaggregation, respectively. 44 h after reaggregation, cell
aggregates were transferred into hydra medium. Homoaggregates were prepared in
each experiment to control the size and the quality of resulting
aggregates.
Tissue labelling with fluorescent beads
Fluorescent latex beads labelled with fluorescein isothiocyanate (FITC)
were used to label hydra cells by phagocytosis as described previously
(Technau and Holstein, 1992).
Endodermal epithelial cells could be selectively labelled by injecting a 2.5%
bead suspension into the gastric cavity using a glass capillary. For selective
labelling of ectodermal epithelial cells, 24 h starved H. vulgaris
polyps were incubated for 15 h in a 0.025% suspension of beads.
Transmission electron microscopy
Polyps were relaxed in 2% urethane prior to fixation in 3.5% glutaraldehyde
in 0.05 mol l-1 cacodylate buffer, pH 7.4, for 18 h at 4°C.
After washing with 0.075 mol l-1 cacodylate buffer for 30 min,
animals were postfixed with 1% OsO4 in 0.075 mol l-1
cacodylate buffer for 2 h at 4°C. After additional washing for 30 min the
tissue was dehydrated in ethanol and embedded in Agar 100 resin (Agar
Scientific, Ltd., Essex). Semithin sections were stained according to
Richardson et al. (1960) with
a solution containing 0.5% Methylene Blue, 0.5% borax, and 0.5% Azur II in
ddH2O at 60°C for 1-2 min. Ultrathin sections were contrasted
with 2.5% uranylacetate and lead citrate solution (prepared freshly from lead
acetate and sodium citrate) for 2 min
(Reynolds, 1963
) and analysed
using a transmission electron microscope CM10 or EM 208 S (Philips).
Immunohistochemistry using species-specific antibodies
Species-specific mouse polyclonal antisera were raised against membrane
fractions of H. vulgaris and H. oligactis
(Samoilovich et al., 2001). To
remove cross-species reacting antibodies, the antiserum was preincubated with
fixed cells of the opposite species overnight at 4°C. For light
microscopy, polyps were fixed with 4% formaldehyde in hydra medium, dehydrated
in ethanol and embedded in histoplast-S (Serva) according to the
manufacturer's protocol. 6 µm sections were prepared, dehydrated in ethanol
and stained with the polyclonal mouse antisera (diluted 1:500) prior to
detection with a FITC-labelled sheep-anti-mouse secondary antibody (Boehringer
Mannheim Biochemica). Sections were then counterstained with Toluidine Blue.
Whole-mount staining was performed as described for paraffin sections. For
immunostaining on ultrathin sections, unspecific binding sites were blocked by
incubating the grids in PBS containing 0.1% Triton and 3% BSA (PBS-Triton-BSA)
for 30 min. Incubation with the primary antibody in PBS-Triton-BSA was done at
37°C for 1 h. Preparations were washed for 4x 5 min with
PBS-Triton-BSA and incubated with the secondary goat-anti-mouse antibodies
(Sigma) coupled with 10 nm large gold particles (1:20 dilution in
PBS-Triton-BSA) for 40 min at 37°C. Preparations were then washed 4x
5 min in PBS-Triton-BSA followed by washing 4x 1 min with
ddH2O. The preparations were contrasted with uranylacetate and lead
citrate as described.
Detection of programmed cell death
Hydra cells undergoing apoptosis were detected as described
previously (Kuznetsov et al.,
2001). Animals were stained in 0.1 µmol l-1 Acridine
Orange, a fluorescent dye that is widely used to specifically highlight
apoptotic cells in a variety of organisms including Hydra
(Cikala et al., 1999
). DNA
fragmentation, as an indicator of apoptosis, was detected by TUNEL [terminal
deoxynucleotidyl transferase-mediated digoxigenin (DIG)-dUTP nick-end
labelling] using the TdT-FragEL DNA fragmentation Kit (Amersham Pharmacia
Biotech) and 1 µmol l-1 DIG-dUTP. Detection of DIG was carried
out by standard procedures using an anti-DIG antibody coupled with alkaline
phosphatase and phosphatase reaction with nitroblue tetrazolium chloride
(NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) as a substrate.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Stability of interspecific grafts
When culturing heterografts we observed that interspecies combinations are
not permanently stable (Fig.
2). H. vulgaris/H. oligactis heterografts were perfectly
healed 24 h after transplantation. However, a few days later, a constriction
appeared at the graft border of the interspecies combinations and a clump of
tissue of unknown origin appeared in the gastric cavity close to the graft
junction (Fig. 3A). Within 3
weeks all of the 179 H. vulgaris/H. oligactis grafts from 5
independent experiments eventually separated, regardless of whether H.
vulgaris was the apical or the basal partner. In contrast, control H.
vulgaris/H. vulgaris and H. oligactis/H. oligactis homografts
formed permanently stable combinations. Since we previously observed
displacement but no separation of the graft partners
(Bosch and David, 1986), these
findings stimulated us to study the cellular interactions in more detail.
|
|
Differential epithelial movement and displacement of H. vulgaris
endoderm
To investigate the nature of the amorphous tissue material accumulating at
the graft site, we performed detailed histological analysis. As shown in
Fig. 3B, the tissue clump
corresponds to endodermal tissue of H. vulgaris detached from the
mesoglea. This indicated that H. oligactis endoderm displaces the
endoderm of H. vulgaris at the graft site. To assess the dynamics of
this tissue displacement, we differentially labelled the endoderm of H.
oligactis using fluorescent beads.
Fig. 3D shows such a
interspecies combination 2 h after grafting with a sharp boundary between
unlabeled H. vulgaris and labeled H. oligactis endoderm. 22
h later fluorescently labelled H. oligactis endoderm is found in the
H. vulgaris portion of the graft
(Fig. 3E). Thus, endoderm of
H. oligactis underwent rapid movement and displaced H.
vulgaris endoderm. Similar tissue movement and displacement of H.
vulgaris endoderm could be observed regardless of whether H.
vulgaris was the apical or basal partner of the heterograft. In control
homografts, neither overlapping nor tissue displacement was observed 24 h
after grafting (data not shown). The displacement of H. vulgaris
endoderm by H. oligactis endoderm leads to the overlap of two
heterogeneous tissue layers at the graft junction (see
Fig. 3C). It seems possible
that this overlap contributes to the observed local induction of apoptosis at
the graft site.
Impaired cellmatrix and cellcell contact at the graft
site
To investigate the role of cell adhesiveness in the observed instability of
the heterografts, cell contacts at the graft site were analysed at the
ultrastructural level. The graft junction of 1-day-old H. vulgaris/H.
oligactis heterografts was identified by both H.
vulgaris-specific antiserum binding to the apical surface of ectodermal
epithelial cells (Fig. 4; see
Materials and methods) and the morphology of the glycocalyx, which is thin in
H. vulgaris and thick in H. oligactis (Figs
4 and
5). As shown in
Fig. 5C, heterotypic cell
contact between an ectodermal epithelial cell of H. oligactis and of
H. vulgaris is characterized by scattered septae-like elements in the
places of desmosomes with the spaces between cell membranes being highly
irregular (Fig. 5C). In
comparison, epithelial contacts in intraspecies grafts are characterized by
well developed septae and regular spaces between cell membranes
(Fig. 5B,D). These observations
suggest that instability of the H. vulgaris/H. oligactis heterografts
might be caused by impaired cellcell contact between heterogeneous
ectodermal epithelial cells.
|
|
The observed overlap of H. vulgaris endoderm and H. oligactis ectoderm at the graft site (Fig. 3B,C) raises the question of the nature and structure of the extracellular matrix separating the two cell layers. To assess this, we studied the extracellular matrix in 24 h old heterografts by electron microscopy. As shown in Fig. 6, distinct morphological differences were observed between the mesoglea at the graft site and outside of it. At the graft site the mesoglea was highly irregular in shape and structure; in some places it appeared as a thick layer (Fig. 6C,F) while in other regions it was flattened (Fig. 6D,E) or even fragmented (Fig. 6C,E). The mesoglea outside the graft region (Fig. 6C,G) was well constituted and showed a structure and thickness similar to that known in normal polyps.
|
Development of heteroaggregates
To monitor cell behavior during heterogenous contact more quantitatively,
we used a cell aggregation assay. Polyps of H. vulgaris and H.
oligactis were dissociated into cell suspensions, combined and aggregated
by centrifugation. The resulting aggregates were allowed to develop. At
different stages of development aggregates were fixed, embedded and sectioned.
To visualize the behavior of cells in the interspecies mixture, we used
immunostaining with species-specific antibodies. As shown in
Fig. 7, starting from a
homogeneous mixture, reaggregated cells from both species quickly became
spatially segregated. That is, ectodermal and endodermal cells sorted into
their respective cell layers in a species-specific manner, resulting in the
formation of clusters of cells of each species 24 h after reaggregation
(Fig. 7C). In agreement with
the behavior observed in heterografts, in most cases, H. vulgaris
ectoderm finally engulfed H. oligactis endoderm
(Fig. 7D,E). Interspecies cell
combinations never remained randomly mixed, indicating that cells from both
species differ in the strengths of their adhesion. Moreover, heteroaggregates
were severely retarded in their development when compared to homoaggregates
(Fig. 7F). Thus, since
development in Hydra is mainly controlled by epithelial cells
(Bosch, 1998), the chimeric
overlapping of epithelia of different species may result in impaired
transduction of developmental signals.
|
When culturing H. oligactis/H. vulgaris heteroaggregates we observed that, in contrast to epithelial cells, cells of the interstitial cell lineage did not sort in a species-specific manner. We used holotrichous isorhiza nematocytes, which differ in their morphology between H. oligactis and H. vulgaris, as a marker for the interstitial cell lineage. 45 days after reaggregation of interspecies cell mixtures, holotrichous isorhiza nematocytes of the H. oligactis type were still found to be homogeneously mixed with those of the H. vulgaris type in tentacles of the developing chimaeras (data not shown). Since nematocytes of both species can also be detected in tentacles of interspecies grafts (Fig. 7G-I), this observation supports the view that a discriminative allorecognition system, if present, is rather poorly developed in Hydra. This is in agreement with observations presented elsewhere (S. G. Kuznetsov and T. C. G. Bosch, manuscript in preparation) that in intraspecies grafts of various H. vulgaris strains, contact to allogeneic tissue did not evoke any response in terms of phagocytosis and elimination of non-self cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis in interspecies grafts in Hydra
Apoptosis functions as part of a quality control process, eliminating cells
that are abnormal, nonfunctional or potentially dangerous to the organisms
(Jacobson et al., 1997). In
Hydra apoptosis has the same morphological features (Bosch and David,
1984
,
1986
) and uses the same
molecular components (Cikala et al.,
1999
) as in more complex animals. Previous studies have shown that
apoptosis in Hydra is involved in elimination of `excess' cells in
response to starvation (Bosch and David,
1984
; Cikala et al.,
1999
) as well as in oogenesis (Miller et al., 2000) and
spermatogenesis (Kuznetsov et al.,
2001
). Recognition and engulfment of apoptotic cells in
Hydra is carried out by ectodermal epithelial cells, which play an
active role as phagocytes (Bosch and David,
1984
,
1986
;
Kuznetsov et al., 2001
). The
present study with Acridine Orange and TUNEL staining confirmed that the cells
phagocytosed by epithelial cells at the graft site underwent apoptosis.
Although the sequence of events leading to activation of apoptosis at the
graft site remains to be elucidated, we present evidence that the loss of cell
anchorage to the extracellular matrix (anoikis) might be a critical step.
Impaired cellmatrix and/or cellcell contact may lead to
induction of programmed cell death
The extracellular matrix separating the two cell layers in Hydra,
termed mesoglea, contains macromolecules such as laminins, collagens, heparan
sulfate proteoglycans and fibronectin-like molecules
(Sarras et al., 1991). These
molecules play essential roles in cell proliferation, cell migration and
morphogenesis (Sarras et al.,
1993
; Schmid et al.,
1999
; reviewed in Sarras and
Deutzmann, 2001
). Curiously, components of Hydra mesoglea
are differentially synthesized by the two epithelia. Hydra laminin
participates in formation of basement membranes of both epithelial layers but
is secreted exclusively by the endoderm
(Sarras et al., 1994
).
Conversely, collagen-I is synthesized in the ectoderm
(Deutzmann et al., 2000
).
Production of collagen-I by ectodermal epithelial cells is preceded by and
dependent on the production of laminin by endodermal epithelial cells
(Sarras and Deutzmann, 2001
).
Our observations indicate that the mesoglea in H. vulgaris/H.
oligactis heterografts underwent significant changes in shape and
structure at the graft site. Thus, incompatible species-specific interactions
between certain extracellular matrix molecules may substantially compromise
the mesoglea structure and provoke programmed cell death via anoikis
at the graft site. Taken together, our findings support the idea that the
mesoglea separating both cell layers plays a key role in controlling survival
of epithelial cells in the basal metazoan Hydra. Therefore, it
appears that the dependence of survival of epithelial cells on anchorage to
extracellular matrix molecules is an ancient feature of epithelial homeostasis
and crucial in metazoan development.
Consistent with this idea, chimeric aggregates showed remarkable
retardation of development (Fig.
7F), similar to aggregates in which biosynthesis of mesogleal
proteins is perturbed (Sarras et al.,
1993). Interestingly, cell interactions in developing
heteroaggregates were similar to the one in heterografts and resulted in
chimeric polyps consisting of H. vulgaris ectoderm and H.
oligactis endoderm. This cell sorting in heteroaggregates is probably
caused by differential cell adhesiveness between the species
(Townes and Holtfreter, 1955
;
Steinberg, 1970
;
Sato-Maeda et al., 1994
).
Since substantial signal exchange between ectodermal and endodermal cells is
occurring during in Hydra morphogenesis
(Kishimoto et al., 1996
), it
seems possible that H. oligactis endoderm outcompetes H.
vulgaris endoderm due to the more effective interaction with the
ectodermal layer, even when the ectoderm comprises H. vulgaris
epithelial cells.
In sum, the present study reveals that interspecific cell contact in Hydra initiates apoptosis at the graft site. However, this largely appears to be the result of impaired cellmatrix and cellcell contact. In contrast to our previous assumption we could not detect any evidence for the ability of Hydra epithelial cells to specifically recognize non-self cells.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bosch, T. C. and David, C. N. (1984). Growth regulation in Hydra: relationship between epithelial cell cycle length and growth rate. Dev. Biol. 104,161 -171.[Medline]
Bosch, T. C. G. and David, C. N. (1986). Immunocompetence in Hydra: epithelial cells recognize self-non-self and react against it. J. Exp. Zool. 238,225 -234.
Bosch, T. C. G. (1998). Hydra. In Cellular and Molecular Basis of Regeneration: From Invertebrates to Humans (ed. P. Ferretti and J. Géraudie). Wiley and Sons Ltd., Sussex.
Buss, L. W. (1982). Somatic cell parasitism and the evolution of somatic tissue compatibility. Proc. Natl. Acad. Sci. USA 79,5337 -5341.[Abstract]
Campbell, R. D. and Bibb, C. (1970). Transplantation in coelenterates. Transplant. Proc. 2, 202-211.[Medline]
Cikala, M., Wilm, B., Hobmayer, E., Bottger, A. and David, C. N. (1999). Identification of caspases and apoptosis in the simple metazoan Hydra. Curr. Biol. 9, 959-962.[Medline]
Deutzmann, R., Fowler, S., Zhang, X., Boone, K., Dexter, S.,
Boot, H., Rachel, R. and Sarras, M. P. (2000). Molecular,
biochemical and functional analysis of a novel and developmentally important
fibrillar collagen (Hcol-I) in hydra. Development
127,4669
-4680.
Frank, U., Leitz, T. and Muller, W. A. (2001). The hydroid Hydractinia: a versatile, informative cnidarian representative. BioEssays 23,963 -971.[Medline]
Frisch, S. M. and Francis, H. (1994). Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol. 124,619 -626.[Abstract]
Fujisawa, T., David, C. N. and Bosch, T. C. (1990). Transplantation stimulates interstitial cell migration in hydra. Dev. Biol. 138,509 -512.[Medline]
Gierer, A., Berking, S., Bode, H., David, C. N., Flick, K., Hansmann, G., Schaller H. and Trenkner, E. (1972). Regeneration of hydra from reaggregated cells. Nature New Biol. 239,98 -101[Medline]
Holstein, T. W., Campbell, R. D. and Tardent, P. (1990). Identity crisis. Nature 346, 21-22.
Jacobson, M. D., Weil, M. and Raff, M. C. (1997). Programmed cell death in animal development. Cell 88,347 -354.[Medline]
Kanaev, I. I. (1969). Hydra. Essays on the Biology of Fresh Water Polyps, pp.1 -452.
Kishimoto, Y., Murate, M. and Sugiyama, T.
(1996). Hydra regeneration from recombined ectodermal and
endodermal tissue. I. Epibolic ectodermal spreading is driven by cell
intercalation. J. Cell Sci.
109,763
-772.
Kolenkine, X. (1958). Evolution des hydres chimères obtenues apres hétérogreffe entre Hydra attenuata et Pelmatohydra oligactis. C. R. Acad. Sci. 246,1748 -1753.
Kuznetsov, S. G., Lyanguzova, M. and Bosch, T. C. G. (2001). Role of epithelial cells and programmed cell death in Hydra spermatogenesis. Zoology 104, 25-31.
Maier, S., Tertilt, C., Chambron, N., Gerauer, K., Huser, N., Heidecke, C. D. and Pfeffer, K. (2001). Inhibition of natural killer cells results in acceptance of cardiac allografts in CD28-/- mice. Nat. Med. 7,557 -562.[Medline]
Meier, P., Finch, A. and Evan, G. (2000). Apoptosis in development. Nature 407,796 -801.[Medline]
Meredith, J. E., Fazeli, B. and Schwartz, M. A. (1993). The extracellular matrix as a cell survival factor. Mol. Biol Cell 4,953 -961.[Abstract]
Miller, M. A. and Steele, R. E. (2000). Lemon encodes an unusual receptor protein-tyrosine kinase expressed during gametogenesis in Hydra. Dev. Biol. 224,286 -298.[Medline]
Mokady, O. and Buss, L. W. (1996). Transmission
genetics of allorecognition in Hydractinia symbiolongicarpus
(Cnidaria:Hydrozoa). Genetics
143,823
-827.
Reynolds, A. S. (1963). The use of lead citrate
at high pH as an electron-opaque stain in electron microscopy. J.
Cell Biol. 17,208
-212.
Richardson, K. C., Jarett, L. and Finke, E. H. (1960). Embedding in epoxy resins for ultrathin sectioning in electron microscopy. Stain Technol. 35,313 -325.
Rinkevich, B. (1996). Immune responsiveness in marine invertebrates revisited: the concourse of puzzles. In New Directions in Invertebrate Immunology (ed. K. Soderhall, K. Soderhull and S. Iwanaga), pp. 55-90. Fair Haven, USA: SOS Publ.
Samoilovich, M. P., Kuznetsov, S. G., Pavlova, M. S. and Klimovich, V. B. (2001). Monoclonal antibodies against antigens of Hydra vulgaris and Hydra oligactis. J. Evol. Biochem. Physiol. 37,262 -269.
Sarras, M. P., Madden, M. E., Zhang, X. M., Gunwar, S., Huff, J. K. and Hudson, B. G. (1991). Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization. Dev. Biol. 148,481 -494.[Medline]
Sarras, M. P., Zhang, X., Huff, J. K., Accavitti, M. A., St John, P. L. and Abrahamson, D. R. (1993). Extracellular matrix (mesoglea) of Hydra vulgaris III. Formation and function during morphogenesis of hydra cell aggregates. Dev. Biol. 157,383 -398.[Medline]
Sarras, M. P., Yan, L., Grens, A., Zhang, X., Agbas, A., Huff, J. K., St John, P. L. and Abrahamson, D. R. (1994). Cloning and biological function of laminin in Hydra vulgaris. Dev. Biol. 164,312 -324.[Medline]
Sarras, M. P. and Deutzmann, R. (2001). Hydra and Niccolo Paganini (1782-1840) two peas in a pod? The molecular basis of extracellular matrix structure in the invertebrate, Hydra.BioEssays 23,716 -724.[Medline]
Sato-Maeda, M., Uchida, M., Graner, F. and Tashiro, H. (1994). Quantitative evaluation of tissue-specific cell adhesion at the level of a single cell pair. Dev. Biol. 162, 77-84.[Medline]
Schmid, V., Ono, S. I. and Reber-Muller, S. (1999). Cell-substrate interactions in cnidaria. Microsc. Res. Tech. 44,254 -268.[Medline]
Steinberg, M. S. (1970). Does differential adhesion govern self-assembly processes in histogenesis? Equilibrium configurations and the emergence of a hierarchy among populations of embryonic cells. J. Exp. Zool. 173,395 -433.[Medline]
Technau, U. and Holstein, T. W. (1992). Cell sorting during the regeneration of Hydra from reaggregated cells. Dev. Biol. 151,117 -127.[Medline]
Townes, P. L. and Holtfreter, J. (1955). Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 128,53 -120.
Weil, M., Jacobson, M. D., Coles, H. S. R., Davies, T. J., Gardener, R. L., Raff, K. D. and Raff, M. C. (1996). Constitutive expression of the machinery for programmed cell death. J. Cell Biol. 133,1053 -1059.[Abstract]