Developmental Biology 248, 199 –219 (2002)
doi:10.1006/dbio.2002.0744
REVIEW
Developmental Signaling in Hydra:
What Does It Take to Build a “Simple” Animal?
Robert E. Steele 1
Department of Biological Chemistry and the Developmental Biology Center,
University of California, Irvine, California 92697-1700
Developmental processes in multicellular animals depend on an array of signal transduction pathways. Studies of model
organisms have identified a number of such pathways and dissected them in detail. However, these model organisms are all
bilaterians. Investigations of the roles of signal transduction pathways in the early-diverging metazoan Hydra have revealed
that a number of the well-known developmental signaling pathways were already in place in the last common ancestor of
Hydra and bilaterians. In addition to these shared pathways, it appears that developmental processes in Hydra make use of
pathways involving a variety of peptides. Such pathways have not yet been identified as developmental regulators in more
recently diverged animals. In this review I will summarize work to date on developmental signaling pathways in Hydra and
discuss the future directions in which such work will need to proceed to realize the potential that lies in this simple
animal. © 2002 Elsevier Science (USA)
“The cnidarians never stop pulling surprises.”
Colin Tudge
The Variety of Life, 2000
INTRODUCTION
We know a lot about the developmental biology of a very
small number of animals. Concentrated efforts on several
model systems (especially those with tractable genetics)
have revealed in marvelous detail the circuitry which
controls the developmental fate of individual cells and
tissues. More recently the question of how organ development is regulated has begun to be approached. By comparisons of the data obtained from studies of animals in diverse
taxa, we can hope to determine the evolutionary routes
taken in the formation of the extant animals. From the data
obtained to date, we are beginning to obtain a picture of
how development in bilaterian animals evolved. However,
a considerable amount of metazoan evolution took place
before bilaterians appeared on the scene. It is by examining
phyla which arose during this initial phase of metazoan
evolution that one expects to find the information that will
1
Fax: (949)824-2688. E-mail: [email protected].
0012-1606/02 $35.00
© 2002 Elsevier Science (USA)
All rights reserved.
tell us how animals were first assembled. Over the past 15
years, Hydra has moved to center stage in such studies.
THE EVOLUTIONARY CONTEXT OF
HYDRA DEVELOPMENTAL BIOLOGY
From molecular phylogenetic studies, it is clear that the
cnidarians, the ctenophores, and the sponges diverged prior
to the appearance of the bilaterians (Adoutte et al., 2000;
Cavalier-Smith et al., 1996; Collins, 1998; Kim et al., 1999;
Medina et al., 2001; Wainright et al., 1993). Thus, comparisons of the molecular features of developmental processes
in these taxa with those in bilaterians have much to say
about what occurred as evolution assembled the organism
that gave rise to modern bilaterians. Among these three
taxa, the cnidarians have been the most intensively studied
at the molecular level. In particular, Hydra has been the
most well-studied due to its long history as an experimental
organism and the ease with which it can be cultured and
manipulated in the laboratory (Bode and Bode, 1984b;
Lenhoff, 1983). One should keep in mind, however, that
Hydra is a highly derived cnidarian (Collins, 2000; Harrison
and Westfall, 1991). It lives in fresh water, while nearly all
other cnidarians are marine. It lacks a free-living larva,
199
200
Robert Steele
which is present in virtually all other members of the class
Hydrozoa. It lacks the medusa stage of the life cycle, which
is found in many hydrozoan taxa. Thus, as is the case for
many other experimental systems, efforts have been put
into an organism which is tractable as a laboratory animal,
not one which is most representative of its phylum. Nonetheless, Hydra clearly has much to tell us about evolutionary solutions to developmental problems in an animal far
removed phylogenetically from those we know so well.
WHAT DOES HYDRA DEVELOPMENT
INVOLVE?
The study of Hydra developmental biology is dominated
by attempts to understand two phenomena. One is the basis
for the establishment and maintenance of the axial pattern
(Bode and Bode, 1984b). In simple terms, this can be phrased
as the question “how does Hydra know to put a head at one
end and a foot at the other?” The other component of the
Hydra developmental equation is the question of how the
various cell lineages in the adult polyp are derived from
their stem cell precursors (Bode, 1996). If one could give
detailed mechanistic descriptions of these two phenomena,
developmental processes in the adult Hydra polyp would in
essence be understood. At this point, it should be made
clear that developmental biologists studying Hydra are in a
peculiar position. They study developmental phenomena in
the adult, although Hydra has perfectly good embryonic
development (Martin et al., 1997). But because of the
developmental nature of the adult polyp and the variety of
ways in which it can be experimentally manipulated, virtually all of the efforts directed at Hydra development have
been directed at the adult. This puts us in the seemingly
awkward position of comparing development in the adult
Hydra polyp to processes occurring in embryos of other
animals. However, as discussed below, the tissue dynamics
of the Hydra polyp require that axial patterning and cell
differentiation occur continuously in the adult animal. As a
result of this situation, it has been possible to use the adult
Hydra polyp to learn a great deal about developmental
processes in cnidarians.
The developmental dynamics of the adult Hydra polyp
have been described in detail in a review by Bode and Bode
(1984b), and they will only be summarized here (Fig. 1). The
adult polyp consists of two epithelial cell layers (the ectoderm and the endoderm) which are concentrically arranged
to form a two-layered tube surrounding the gastric cavity.
At the two ends of the tube are, respectively, a dome (the
hypostome) containing the mouth opening surrounded by a
ring of tentacles (which together constitute the head) and a
disk of cells that allows the animal to adhere to the
substratum (the foot or basal disk). All of the epithelial cells
of the body column (the part of the animal exclusive of the
head and foot) are mitotically active (Holstein et al., 1991).
The cells in the tentacles and the foot are arrested in the G 2
phase of the cell cycle (Dübel et al., 1987). The animal
FIG. 1. Tissue dynamics and cell division in the adult Hydra
polyp. This figure is adapted from Campbell (1967b) and shows the
results of a study in which tissue movements were followed by
marking the polyp with methylene blue or grafted tentacles. The
arrows indicate the starting and ending positions of the marked
tissue, and the number of days required for transit of the marked
tissue are indicated. Blue arrows indicate the paths of marked
tissue which moved toward the head, red arrows indicate the paths
of marked tissue which moved toward the foot or onto buds, and
the green bracket indicates the region in which marked tissue
showed little or no movement. The portion of the polyp colored
yellow is the region in which epithelial cells are dividing (Campbell, 1967a; Holstein et al., 1991). The regions in turquoise (tentacles and basal disk) are where cells are arrested in G 2 of the cell
cycle (Dübel et al., 1987).
maintains its fixed adult size in the face of continued
production of cells in the body column by the shunting of
cells into buds, the asexual form of reproduction, and by
loss of cells through sloughing from the distal ends of the
tentacles and from the foot (Campbell, 1967b, 1973). These
tissue dynamics present the Hydra polyp with one of its
two major developmental problems. Due to the production
and loss of cells, an epithelial cell in the body column is
continually changing its axial position. Thus, an epithelial
cell that is mitotically active and located in the body
column will eventually find itself crossing either into the
© 2002 Elsevier Science (USA). All rights reserved.
201
Developmental Signaling in Hydra
head or the foot, where it must arrest the cell cycle and
differentiate into a new type of cell, either a tentacle battery
cell or a foot basal disk cell. This process is even more
dramatically brought about when a Hydra is bisected
through the body column. In such a case, cells located in
the middle of the animal suddenly find themselves at the
ends and must adopt the appropriate developmental fates to
allow them to regenerate the missing structures (head and
foot). It seems clear, then, that Hydra cells must have
signaling pathways which allow them to sense their location in the animal with a high degree of accuracy and that
such sensing must be coupled to pathways which regulate
developmental fate decisions.
The other developmental process in Hydra in which cell
fates must be specified is the differentiation of the various
products of the interstitial cell compartment (Bode, 1996;
Campbell and David, 1974). The interstitial cell compartment in Hydra functions much like the bone marrow of a
vertebrate. This compartment (Fig. 2) consists of multipotent stem cells which give rise to four classes of differentiation products—nerves, gametes, secretory cells, and
nematocytes (the “stinging” cells used for prey capture).
Within the gamete lineages, there are committed stem
cells, one for eggs and one for sperm (Littlefield, 1985, 1991,
1994). Homeostasis within the interstitial cell compartment almost certainly requires communication between
the cells of the compartment and the epithelial cells via
signaling pathways. As is the case with the epithelial cells,
cells within the interstitial cell compartment must also
sense axial position. The nervous system of Hydra consists
of a variety of nerve types, which express particular genes
(e.g., those encoding neuropeptides) dependent on their
position within the animal (Bode, 1992). For example, there
are nerves which don’t express a particular neuropeptide
while in the body column but which begin expressing it
when they move into the head via the tissue displacement
process described above (Koizumi and Bode, 1986). Thus,
nerves must also have a mechanism for sensing their
position in the animal.
Given the clear necessity that cells in the adult Hydra
polyp receive extracellular signaling inputs in order to
adopt and maintain the appropriate developmental fates,
what molecules are there that might provide such inputs?
Three approaches have been used to search for such molecules. Initial efforts used biological assays to identify
activities which affected developmental processes in Hydra, for example, activities which accelerated regeneration
of the head or the foot. Although this approach was arduous
because of the large amounts of materials required to purify
the active components and the relatively subtle nature of
the response, three molecules were purified to homogeneity
by using this approach. The first molecule identified was
the so-called head activator (HA) (Schaller and Bodenmüller, 1981), a peptide which was identified by its ability to
increase the rate of head regeneration when applied to
decapitated Hydra polyps (Schaller, 1973). In addition, two
peptides have been identified by virtue of their ability to
accelerate foot regeneration (Hoffmeister, 1996).
The second approach taken to identify signaling pathway
components in Hydra has involved treating the animal
with compounds which are known to perturb signaling
pathways in other systems. The most successful experiments in this regard involved treatment with diacylglycerol
(DAG) (Müller, 1989) or lithium chloride (Hassel et al.,
1993; Hassel and Berking, 1990). As discussed below, these
compounds have dramatic effects on patterning processes
in the Hydra polyp.
Finally, efforts to clone and characterize genes from
Hydra were begun in the late ’80s, and these efforts have
resulted in the identification of a number of genes for which
there is evidence of a role in developmental signaling
(Galliot, 2000). In the following sections, I will discuss the
results which have been gained from all three of these
approaches. A summary of the developmental signaling
pathways and their component molecules identified in
Hydra is presented in Table 1.
HEAD ACTIVATOR, THE FIRST
DEVELOPMENTAL SIGNALING
MOLECULE IDENTIFIED IN HYDRA
In 1973, Chica Schaller identified a protease-sensitive
activity in extracts of Hydra which accelerated regeneration of the head but not the foot (Schaller, 1973), which she
termed head activator. While HA accelerates head regeneration, as measured by counting the number of tentacles
which appear 48 h following head removal (Schaller et al.,
1983), it does not affect the final number of tentacles when
regeneration is complete (Javois and Tombe, 1991). HA
activity was also found in extracts from another cnidarian,
the sea anemone Anthopleura elegantissima. The complete
sequence of the sea anemone head activator was determined, and it was found to be an 11-amino-acid peptide
(Schaller and Bodenmüller, 1981). While the Hydra head
activator peptide has never been sequenced, proteolytic
fragments from Hydra HA have identical amino acid compositions to the corresponding fragments from sea anemone
HA (Schaller and Bodenmüller, 1981). HA is difficult to
work with due to its propensity for forming inactive dimers
in solution (Bodenmuller et al., 1986), and efforts by several
labs to clone the gene encoding it have been unsuccessful.
In addition, HA has not yet been reported to be among the
peptides sequenced by the Hydra Peptide Project (see below). Using the head regeneration bioassay, Schaller and
Gierer (1973) found that the specific activity of HA varied
along the oral/aboral axis of the polyp, being highest in the
oral quarter of the animal and lowest in the aboral quarter
of the animal. Fractionation studies localized the biological
activity to a fraction containing membraneous particles
which were hypothesized to be derived from the Golgi/
endoplasmic reticulum compartment (Schaller and Gierer,
1973). Surprisingly, fixed cells retain HA activity and den-
© 2002 Elsevier Science (USA). All rights reserved.
202
Robert Steele
sity gradient centrifugation of cells from fixed, dissociated
Hydra polyps showed that the activity was enriched in the
fraction enriched for nerve cells (Schaller and Gierer, 1973).
Attempts to examine the distribution of HA in Hydra
tissue by immunocytochemistry (Schawaller et al., 1988)
have yielded results which are not easily reconciled with
the earlier data from cell fractionation studies. Monoclonal
antibodies were raised against HA peptides coupled to
keyhole limpet hemocyanin. While such antibodies stain
Hydra cells, the staining was primarily in interstitial cells.
Nerve cells were not stained. Interestingly, interstitial cells
in the head region of buds were heavily stained. Cloning of
the HA gene will allow a revisiting of these observations
and should help a great deal in further dissecting the biology
of HA.
If HA is required for formation and maintenance of the
head, its apparent location in nerve cells would predict that
nerve-free Hydra should have defects in head formation. It
is possible to produce nerve-free Hydra by various treatments that selectively eliminate the stem cells from the
interstitial cell compartment (Lenhoff, 1983). Depending on
the type of treatment used to produce them, such nerve-free
animals (so-called epithelial Hydra) can be maintained
indefinitely by hand feeding (Marcum, 1983), since they are
unable to capture and ingest prey themselves. Surprisingly,
nerve-free Hydra maintain their normal axial pattern, bud,
and regenerate (Marcum and Campbell, 1978). Analysis of
extracts of nerve-free Hydra revealed that the animals
retained HA activity and that, in fact, the specific activity
in such animals was eight times that seen in normal
animals (Schaller et al., 1980). As suggested by Schaller et
al. (1980), this result may indicate a latent capacity of the
epithelial cells to produce HA, a capacity which is normally
blocked by the nerve cells.
Subsequent to identifying the HA effect on head regeneration, Schaller (1976a) demonstrated that Hydra treated
with HA showed an increase in cell division in both the
epithelial cell compartment and the interstitial cell compartment, indicating that HA is a mitogen. Analysis of
tentacle regeneration in HA-treated polyps has demonstrated that the acceleration of tentacle regeneration seen in
such polyps was due to the generation of a larger pool of
tentacle cells during regeneration via the mitogenic activity
of HA (Hobmayer et al., 1997).
In addition to its effects on head development in the
regenerating and normal polyp, HA has been shown to
stimulate budding (Hobmayer et al., 1997; Schaller, 1973),
Hydra’s asexual mode of reproduction. Schaller (1973)
treated animals with one bud for 24 h with HA and then
counted the number of animals which had initiated a
second bud by the end of the 24-h treatment. Among
untreated control animals, 30% had initiated a second bud
at 24 h. For the HA-treated animals, 50% had initiated a
second bud at 24 h. Hobmayer et al. (1997) examined the
effect on budding of longer-term exposure to HA. They
collected newly dropped buds and fed them until the first
bud began to form. At this point, feeding was stopped and
the animals were exposed to HA for 4 days. Control
polyps were treated identically, except that they were not
exposed to HA. The HA-treated polyps had formed close
to two buds per animal after the 4-day treatment,
whereas untreated controls had formed only one bud.
Interestingly, the number of epithelial cells did not
increase significantly during the 4-day period in either
the controls or the HA-treated animals. Thus, the extra
bud formed by the HA-treated animals was not due to HA
stimulation of cell division.
Finally, various studies have also indicated that HA has
effects on the interstitial cell compartment. During head
regeneration, about 450 new nerve cells must be formed in
a period of 48 h (Bode et al., 1973). Labeling with tritiated
thymidine showed that these new nerves arise from dividing precursor cells (Schaller, 1976b). Treatment of regenerating polyps with HA stimulates the formation of new
nerves during head regeneration to an even higher degree
than is seen in untreated animals (Schaller, 1976b). These
results were taken to indicate that HA acts to increase the
determination of interstitial cells to become nerve cells.
However, additional studies have indicated that the role of
HA in the nerve cell developmental pathway is likely to be
more complicated than this (Hobmayer et al., 1990;
Hoffmeister and Schaller, 1987; Holstein and David, 1986;
Holstein et al., 1986). Until molecular and functional
approaches can be used to more precisely define the effects
of HA on the interstitial cell lineage, it will remain unclear
exactly how HA acts in this lineage.
Until recently, HA stood in isolation, with no indication
of the nature of the signaling pathway by which it acts.
Given that HA is a neuropeptide, a G protein-coupled
seven-transmembrane molecule seemed a good bet for its
receptor. An HA-binding protein with a K d of about 1 nM
has been identified (Franke et al., 1997). Surprisingly, this
binding protein is a member of the low-density lipoprotein
(LDL) receptor superfamily (Hampe et al., 1999); in particular, it is the homologue of a mammalian protein called
sorLA (Jacobsen et al., 1996). This was unexpected, particularly since there is no evidence that sorLA has a signal
transduction role (Jacobsen et al., 2001), although recently,
other members of the LDL receptor superfamily have been
shown to have signal-transducing capacity (Herz, 2001;
Howell and Herz, 2001). Of particular interest with regard
to developmental events is the finding that LRP6, a member
of this superfamily, acts together with Frizzled as a coreceptor for Wnt (Pinson et al., 2000; Tamai et al., 2000;
Wehrli et al., 2000) and that LRP6 is the receptor for the
dickkopf family of Wnt inhibitors (Mao et al., 2001; Semenov et al., 2001). Since the Wnt pathway is present in
Hydra (see below), the involvement of the HA receptor in
this pathway is an intriguing possibility.
In situ hybridization analysis with the cloned Hydra
sorLA gene demonstrated high levels of expression in the
epithelial cells at the bases of the tentacles and a lower level
of expression throughout the rest of the polyp (Hampe et al.,
1999). Immunostaining of cells from dissociated Hydra
© 2002 Elsevier Science (USA). All rights reserved.
203
Developmental Signaling in Hydra
FIG. 2. Cell lineage compartments in Hydra. This figure shows the three cell lineage compartments in the adult Hydra polyp. The
epithelial cells of the body column consist of the ectodermal and endodermal epithelial stem cells. Thus, the body column is made up
entirely of stem cells which give rise to new body column epithelial cells by division and to the terminally differentiated cells of the foot
and the tentacles following displacement from the body column as shown in Fig. 1. Dividing committed cells of the nerve, nematocyte, and
secretory cell pathways (Bode, 1996) are not shown.
with an antibody against the sorLA protein indicated that
the protein is present in both ectodermal and endodermal
epithelial cells, in all of the precursor cells in the interstitial
cell lineage, and in differentiated nerves (Hampe et al.,
1999). This result is surprising since labeling of interstitial
cells and nerves is not seen with in situ hybridization. In
addition to its transmembrane form, the Hydra sorLA
protein exists in a form which does not sediment at
100,000g and which appears to lack the transmembrane and
cytoplasmic segments (Hampe et al., 1999). The function of
this soluble form is unknown.
Efforts to elucidate the downstream components of the
HA pathway have demonstrated that a 10-min exposure of
intact polyps to HA results in a 2-fold increase in the cyclic
AMP level (Galliot et al., 1995). Polyps exposed for 30 min
show cAMP levels equal to those of control animals,
indicating that the rise in cAMP in response to HA is
transient. Cyclic AMP response element binding (CREB)
activity has been identified in Hydra, and a cDNA encoding
a Hydra CREB protein has been isolated (Galliot et al.,
1995). Electrophoretic mobility shift assays show that
CREB activity rises during the early stages (4 h) of both head
and foot regeneration in Hydra vulgaris and then returns to
the basal level by 28 h. Hydra CREB is a substrate for
protein kinase A in vitro (B. Galliot, personal communication), suggesting that HA could modify CREB protein activity as a result of its effect on the cAMP level.
THE FOOT END
In parallel with the studies in the Schaller lab on head
activator, an activity which accelerated foot regeneration
but not head regeneration was identified in Hydra extracts
(Grimmelikhuijzen and Schaller, 1977). The foot activator
(FA) activity was distributed in a graded manner in the adult
© 2002 Elsevier Science (USA). All rights reserved.
204
Robert Steele
TABLE 1
Components identified in Hydra
Signaling
pathway
Ligand
Receptor
sorLA
Additional
components
head activator
head activator
CREB protein
Pedibin/Hym-346
pedibin/Hym-346
?
?
Pedin
HEADY
Hym-323
Hym-33H
pedin
HEADY
Hym-323
Hym-33H
?
?
?
?
?
?
?
?
Hym-355
Hym-355
?
?
Wnt
Wnt
TGF-␤
BMPs
?
Hedgehog
Hedgehog
?
Frizzled
nuclear receptor
insulin
?
?
COUP
insulin-related
receptor
Shin Guard
?
shin guard
CCK-4/Klg/Dtrk
Notch
?
?
Lemon
?
Endothelin
?
?
?
Integrin/ECM
? (endothelin
converting
enzyme
identified)
?
?
?
extracellular
matrix
disheveled, ␤catenin, GSK3,
Tcf/Lef
chordin, R-Smad
Ci/Gli family
transcription
factor
?
?
?
?
?
Homologues of
Suppressor of
Hairless, Numb,
and hairy/
Enhancer of
Split
?
?
?
Src
protein kinase C
?
integrin
Ras
?
Developmental
roles
head formation,
budding, interstitial
cell development
foot formation
foot formation
head formation
foot formation
nerve cell
differentiation
nerve cell
differentiation
axial patterning
?
?
References
Galliot et al., 1995,
Hampe et al., 1999;
Schaller et al., 1989
Grens et al., 1999;
Hoffmeister, 1996
Hoffmeister, 1996
Lohmann and Bosch, 2000
Harafuji et al., 2001
Takahashi et al., 1997
Takahashi et al., 2000
Hobmayer et al., 2000;
Minobe et al., 2000
B. Reinhardt and H. Bode,
personal communication;
B. Hobmayer,
F. Rentzsch, and
T. Holstein, personal
communication
Kaloulis, 2000
?
cell division, tentacle
and foot cell
differentiation
peduncle formation
and/or foot
formation?
gametogenesis, budding
?
Escriva et al., 1997
Steele et al., 1996
foot development
Zhang et al., 2001
head formation
head and foot
development
head development
head and foot
development
Cardenas et al., 2000
Hassel, 1998; Hassel et al.,
1998; Müller, 1989
Bosch et al., 1995
Deutzmann et al., 2000;
Fowler et al., 2000;
Leontovich et al., 2000;
Sarras and Deutzmann,
2001; Sarras et al., 1991,
1993, 1994; Zhang
et al., 1994
Bridge et al., 2000
Miller and Steele, 2000
P. Towb and J. Posakony,
personal communication
Note. The table lists all of the cases in which at least one component of a ligand-activated signaling pathway that is likely to play a
developmental role has been identified in Hydra. In some cases, there is experimental evidence for a developmental role (e.g., head
activator). In other cases, such a role is assumed based on results from other organisms.
© 2002 Elsevier Science (USA). All rights reserved.
205
Developmental Signaling in Hydra
polyp, being highest at the foot end and lowest at the head
end (Grimmelikhuijzen and Schaller, 1977). Further characterization of FA (Grimmelikhuijzen, 1979) revealed that
it was sensitive to protease, had a low molecular weight,
and was enriched in a nerve cell fraction isolated from
dissociated Hydra by differential centrifugation.
Hoffmeister (1989) developed methods for fractionation
of FA-containing extracts which resulted in an enrichment
of 10 5- to 10 6-fold relative to the starting extract. Using FA
prepared with this approach, Hoffmeister (1989) found a
surprising range of effects. In addition to accelerating foot
regeneration, FA caused a modest stimulation of epithelial
cell division, an increase in division of interstitial cells, and
an increase in the production of nerve cells.
Studies of FA have progressed significantly recently with
the demonstration that the active fraction consists of two
peptides, termed pedin and pedibin (Hoffmeister, 1996). The
Hydra Peptide Project (see below) has identified a peptide
termed Hym-346 which is nearly identical to pedibin,
differing only in the absence of a C-terminal glutamic acid
residue (Takahashi et al., 1997). This difference is apparently due to the fact that pedibin was isolated from Hydra
vulgaris and Hym-346 was isolated from Hydra magnipapillata. Both pedin and pedibin/Hym-346 have similar specific activities with regard to acceleration of foot regeneration (Hoffmeister, 1996). Using a radioimmune assay, the
levels of pedin and pedibin/Hym-346 were determined in
upper and lower halves of bisected polyps (Hoffmeister,
1996). The level of pedibin was higher in the upper half by
a factor of 1.5. The level of pedin was 2-fold higher in the
lower half than in the upper half. Treatment of Hydra
oligactis polyps with pedin stimulated the division of
interstitial cells and increased the ratio of nerve cells to
epithelial cells (Hoffmeister, 1996).
Recent studies on pedibin/Hym-346 have shown that it
can modify the axial patterning process. Exposure of adult
polyps to pedibin or Hym-346 for 5–15 days increases the
fraction of the body column expressing CnNK-2, a homeobox gene expressed in a graded fashion in the endoderm
at the aboral end of the polyp (Grens et al., 1999). Treatment with Hym-346 also significantly increases the ability
of segments from the upper part of the body column to
induce a secondary foot when transplanted into host polyps
(Grens et al., 1999). Aggregates of cells from dissociated
Hydra polyps regenerate into polyps (Gierer et al., 1972).
Heads form first in the aggregates, followed by foot formation. Aggregates were treated with Hym-346 to determine
whether the peptide would effect the course of foot formation (Gierer et al., 1972). In a sample of 25 control aggregates, 31 heads and 1 foot were present after 3 days. In a
sample of 27 Hym-346-treated aggregates, 4 heads and 49
feet were present after 3 days. This result provides dramatic
support for the hypothesis that Hym-346 plays a role in foot
formation.
Molecular analyses of pedibin have recently become
possible as a result of the cloning of a cDNA which encodes
this peptide (Hoffmeister-Ullerich, 2001). Two surprising
findings have emerged from examination of the sequence
and expression of the pedibin gene. First, the predicted
sequence of the precursor polypeptide that yields mature
pedibin lacks a signal peptide. This suggests that secretion
of the peptide occurs via a nontraditional route. In situ
hybridization with a pedibin probe shows a high level of
expression in the endoderm of the peduncle (the region of
the body column between the basal disk and the budding
zone) and surprisingly in the endoderm in the proximal
portions of the tentacles. Given the role of pedibin in foot
formation, the expression in the tentacles is unexpected.
Further studies investigating a possible role of pedibin in
tentacle development are obviously warranted.
HEADY
The Bosch laboratory at the University of Kiel has pioneered the use of differential display PCR to identify genes
whose expression levels change in concert with developmental changes in the Hydra polyp (Bosch and Lohmann,
1998). This approach has the potential for rapidly identifying genes involved in Hydra development without the
requirement for any knowledge of the products encoded by
these genes. Detailed studies on one gene identified using
this approach, a gene termed HEADY, have been published
(Lohmann and Bosch, 2000); these studies provide a striking
validation of the differential display PCR approach for
identifying developmental signaling molecules in Hydra.
A HEADY cDNA clone was isolated from a differential
display PCR screen of transcripts from aggregates of Hydra
cells at various times after aggregation. The sequence of the
clone predicts that the mature HEADY peptide is 12 amino
acids in length and that it arises by proteolytic cleavage and
C-terminal amidation of a 23-amino-acid precursor. The
HEADY gene is expressed at a very low level in intact adult
polyps, but expression is dramatically up-regulated 6 h after
the head is removed. Expression is restricted to a small
portion of the endodermal epithelium at the apex of the
polyp. By 8 h after decapitation, HEADY expression has
dropped significantly. No increase in the level of HEADY
RNA is associated with foot regeneration, indicating that
induction of HEADY gene expression is not simply a
response to wounding of the tissue. While the expression
level of the HEADY gene is very low in the adult head, the
gene is expressed at elevated levels in the developing bud.
Expression is seen throughout the endoderm in early buds,
but becomes restricted to the apical endoderm as the head
structures begin to form. Shortly after the bud detaches
from the parent, the HEADY RNA level drops to that seen
in the adult polyp.
Using an antibody against the predicted HEADY peptide
product, the distribution of the peptide has been examined
(Lohmann and Bosch, 2000). The antibody gives a punctate
staining pattern in the endodermal epithelium, with higher
levels of staining in the body column than in the head and
foot. The stained bodies were interpreted as cytoplasmic
© 2002 Elsevier Science (USA). All rights reserved.
206
Robert Steele
vesicles in which the peptide is stored prior to release from
the cell. Surprisingly, staining is not seen in early buds, and
it was not reported when staining first appears in the bud.
The absence of staining in the early bud, despite the
presence of HEADY RNA, was interpreted as indicating
immediate release of the peptide rather than the vesicular
storage seen in the adult polyp.
RNA interference (RNAi) has been used to inhibit expression of several genes in Hydra, including HEADY (Lohmann and Bosch, 2000; Lohmann et al., 1999; Smith et al.,
2000). Double-stranded HEADY RNA was introduced into
polyps by electroporation, and the animals were decapitated
4 days later. Head regeneration was delayed by 2 days in the
experimental animals relative to animals electroporated in
the absence of RNA, indicating that HEADY gene expression is required for head regeneration.
Two other functional tests have been carried out with
HEADY. Synthetic HEADY peptide was applied to newly
detached buds for a period of 12 days. Budding of the treated
animals began about 1.5 days earlier than the untreated
controls. Animals treated with HEADY peptide for 5 days
were used as donors for grafts to untreated animals. The tissue
from the control animals induced secondary heads in the
hosts in 10% of the cases, whereas tissue from the treated
animals induced secondary heads in 30% of the cases.
The data on HEADY suggest that this peptide plays an
important role in the formation of the Hydra head. How it
plays that role is still far from clear. Knowledge of the
identity and distribution of the receptor for HEADY should
help a great deal in elucidating the pathway by which
HEADY acts. The results from staining of polyps and buds
with the HEADY antibody suggests that the regulation of
HEADY production and release may be quite complex. The
use of a heterologous expression system in which protein
trafficking can be more easily investigated than in Hydra
may be valuable in dissecting these processes. Finally, it
will be of obvious interest to examine the expression of
HEADY during embryogeneis to see whether it has a role in
the initial formation of the head.
THE HYDRA PEPTIDE PROJECT: WHAT
IS HYDRA DOING WITH SO
MANY PEPTIDES?
Following up on the pioneering work of the Schaller lab
on Hydra peptides, an international consortium of researchers initiated, in 1993, a systematic and large-scale
effort to isolate peptides from Hydra and determine
whether they had developmental roles (Bosch and Fujisawa, 2001; Takahashi et al., 1997). This effort, termed
The Hydra Peptide Project, has to date resulted in the
purification of over 800 peptides from adult Hydra polyps
(Bosch and Fujisawa, 2001). This is obviously a remarkable number given Hydra’s apparent simplicity. Once a
peptide has been purified and its sequence determined
(which has been accomplished for 40 of the peptides;
Bosch and Fujisawa, 2001), its biological activity is tested
by using a variety of assays. These assays include testing
effects on cell division, analysis of effects on gene expression using differential display PCR, and analysis of effects on morphogenesis (e.g. budding) (Bosch and Fujisawa, 2001; Takahashi et al., 1997).
To date, three peptides have been reported to have
effects on developmental processes in Hydra. Hym-323, a
16-amino-acid peptide, is apparently derived from a 62amino precursor by proteolytic cleavage (Harafuji et al.,
2001). The precursor lacks a classical signal peptide
sequence, so the route it takes out of the cell is unknown.
In situ hybridzation and antibody staining have been
used to examine the expression pattern of Hym-323
(Harafuji et al., 2001). In situ hybridization detects Hym323 RNA in both the ectodermal and endodermal epithelial cells of the body column; no RNA is detected in the
tentacles or the basal disk. An anti-Hym-323 antibody
stains the body column in a pattern similar to that seen
by in situ hybridization and fails to stain the foot.
However, the antibody shows strong staining of the
tentacles, where no Hym-323 RNA was detected. This
finding is unexpected and suggests the possibility that
Hym-323 RNA decays as cells are displaced from the
body column into the tentacles but that the Hym-323
peptide does not turn over in the tentacle cells.
Treatment of polyps with Hym-323 peptide followed by
measurements of the labeling indices of cells in both the
epithelial and interstitial cell compartments showed that
the peptide had no effect on cell cycle behavior in either of
the compartments (Harafuji et al., 2001). Treatment of
regenerating animals with Hym-323 peptide revealed an
acceleration of foot regeneration, but no effect on the rate of
head regeneration. To determine whether the target cells
for the Hym-323 effect were in the epithelial cell compartment or the interstitial cell compartment, polyps were
treated for a short term with colchicine, which kills the
interstitial cell population but spares the epithelial cells
(Campbell, 1976). Hym-323 treatment accelerated foot regeneration in animals depleted of interstitial cells, indicating that the target of the peptide is in the epithelial cell
compartment.
Additional evidence that Hym-323 is part of the foot
development pathway comes from grafting studies with
tissue from Hym-323-treated polyps (Harafuji et al., 2001).
Polyps treated for 3–7 days with Hym-323 were used as graft
donors. Peptide-treated tissue induced a secondary foot in
the host animal in a higher percentage of the cases than did
grafts of untreated tissue.
To determine whether Hym-323 and Hym-346 act
synergistically on foot formation, the rate of foot regeneration was examined in polyps treated with each peptide
alone and the two peptides in combination. No synergism
was detected when the two peptides were used together
(Harafuji et al., 2001). Harafuji et al. (2001) hypothesize
that epithelial cells in the body column release Hym-323
peptide as they are displaced into the foot and differen-
© 2002 Elsevier Science (USA). All rights reserved.
207
Developmental Signaling in Hydra
tiate into basal disk cells. In this scenario, the cells
releasing the peptide would also be the targets, suggesting that Hym-323 acts in an autocrine fashion.
Two peptides, Hym-33H and Hym-355, have been found to
effect nerve cell development in Hydra. Hym-33H, a fiveamino-acid peptide with the sequence AALPW, was originally
shown to reduce specifically the labeling of nerve cells in
polyps pulse-labeled with bromodeoxyuridine (Takahashi et
al., 1997). This finding suggested that Hym-33H inhibits the
progression of cells in the nerve cell differentiation pathway.
The second peptide shown to affect nerve cell differentiation is Hym-355 (Takahashi et al., 2000). A cDNA for
Hym-355 has been cloned, and its predicted protein product
suggests that the Hym-355 precursor polypeptide is secreted by using a classical signal peptide. Proteolytic processing and C-terminal amidation would give rise to the
mature nine-amino-acid peptide. In situ hybridization analysis shows expression of the Hym-355 gene in a subset of
the nerve cell population, with the expressing cells being
found at low density in the body column, and at higher
density in the tentacles and the foot (Takahashi et al.,
2000). This expression pattern has been confirmed by immunostaining with an anti-Hym-355 antibody.
Bromodexoyuridine labeling studies have shown that
Hym-355 does not affect the proliferation of cells in either
the epithelial cell or the interstitial cell compartment
(Takahashi et al., 2000). However, pulse labeling in the
presence of Hym-355 peptide followed by incubation in the
peptide for an additional 47 h results in a 1.5-fold increase
in the number of labeled nerve cells relative to what is seen
in animals not exposed to peptide. Takahashi et al. (2000)
suggest that this result is due to Hym-355 causing either a
decrease in the length of S-phase or an increase in the size
of the nerve precursor cell pool.
Coupling the inhibitory effect of Hym-33H (Takahashi et
al., 1997) with the stimulatory effect of Hym-355 provides
a potential mechanism for maintaining homeostatis in the
nerve cell population of the adult Hydra polyp. If such a
mechanism were operating, one would predict that treatment with a combination of the two peptides should cause
nerve production to occur at the same level as seen in
untreated animals. This turns out to be the case. Thus, it
appears that the level of nerve cell production seen in a
normal adult polyp is established by a balance between a
positive feedback signal from nerves onto nerve cell precursors and an inhibitory signal from epithelial cells onto these
same cells. These findings predict that the receptors for
Hym-33H and Hym-355 will be expressed on the nerve
precursor cell population.
THE WNT PATHWAY: SIGNALING
AT THE SUMMIT
The Wnt signaling pathway is deployed in a large number
of developmental settings in bilaterians (Wodarz and Nusse,
1998). Of particular interest is the role of the Wnt pathway
in establishing the dorsal–ventral axis during embryonic
development (Niehrs, 1999). Thus, it was of considerable
interest to know whether evolution made this pathway
available early enough for it to be used by Hydra. In a
remarkable effort, Hobmayer et al. (2000) succeeded in
cloning the genes for Wnt, dishevelled, GSK3, ␤-catenin,
and Tcf/Lef, nearly all the components of the Wnt signaling
pathway. The Sarras lab (Minobe et al., 2000) has identified
a Frizzled homologue which is the candidate Wnt receptor
in Hydra; Southern blotting indicates that Hydra contains
only a single Frizzled gene. The expression patterns of the
components of the Wnt pathway in Hydra are intriguing.
The Frizzled gene is expressed strongly throughout the
endodermal epithelium of the body column and the foot.
Expression is much lower in the endoderm of the tentacles
and the hypostome. Expression is also much lower in the
ectodermal epithelium. The simplest interpretation of
these data is that the endodermal epithelium is the primary
target of Wnt action and that the specificity of Wnt signaling is achieved by restricting distribution of the ligand or
some other component of the pathway. In support of this
idea are the data on the expression of the Wnt gene
(Hobmayer et al., 2000). Wnt is expressed in a small number
of epithelial cells at the tip of the hypostome in both the
ectoderm and the endoderm. This is the region of the Hydra
polyp which has been shown by transplantation studies to
behave as an axial organizer (Broun and Bode, 2002; Yao,
1945).
The restoration of Wnt expression during head regeneration also fits with Wnt being high up in the axial patterning
hierarchy. Expression begins by 1 h after decaptitation
(Hobmayer et al., 2000). This is to be compared with the
30 –36 h required for complete regeneration of the head in
this study. Perhaps the most rigorous test of whether the
expression pattern of a gene is consistent with a role in axis
formation in Hydra is to examine the pattern in aggregates.
In an aggregate, the axis has been completely disorganized
as a result of the random mixing of the cells. Wnt expression is absent in newly formed aggregates but becomes
detectable in clusters of 10 –20 cells at 24 h following
aggregate formation (Hobmayer et al., 2000). These clusters
ultimately become located at the tips of the hypostomes of
the polyps which form from the aggregates.
The Wnt story in Hydra is, however, not as simple at it
appears from examining the expression patterns of Wnt and
Frizzled. In a nonbudding adult polyp, Tcf/Lef expression is
restricted to the hypostome and the budding region of the
body column. The Tcf/Lef expression domain in the hypostome is somewhat broader than the Wnt domain, and
expression appears to be graded, decreasing as it moves
away from the tip of the hypostome (Hobmayer et al., 2000).
The disheveled, GSK-3, and ␤-catenin genes are expressed
at low and uniform levels throughout the nonbudding adult
polyp. During budding, expression of Tcf/Lef and ␤-catenin
rise in a band around the polyp at the site where the bud
will emerge. Expression of ␤-catenin drops when the head of
the bud begins to develop, and expression of Tcf/Lef be-
© 2002 Elsevier Science (USA). All rights reserved.
208
Robert Steele
comes restricted to the bud head. Wnt expression starts as
the bud tissue begins to evaginate from the parent’s body
column.
Things get even more complicated when one examines
the spatial details of the Wnt expression pattern during
budding and regeneration (B. Hobmayer, personal communication). Wnt expression is restricted to the ectodermal
epithelium of the forming bud but later also becomes
expressed in the underlying endoderm as is the case in the
adult polyp. However, during head regeneration and regeneration of polyps from aggregates, Wnt expression begins in
the endodermal epithelium and then later begins being
expressed in the ectodermal epithelium. The significance of
these differences is at present unknown.
The results of studies to date on the Wnt pathway in Hydra
lead one to conclude that this pathway is a primary component of the axis-forming circuitry of the polyp. At the same
time, the details of the expression patterns of the genes
encoding the pathway components indicate that further dissection of the pathway is not going to be simple. It has been
possible to make soluble Wnt proteins using various cell lines
(http://www.stanford.edu/⬃rnusse/assays/wntproteins.html),
suggesting that it may also be possible to make soluble Hydra
Wnt for experimental tests. The fact that Hydra consists of
only two epithelial layers should facilitate getting the protein
to the target cells, perhaps aided by the addition of dimethylsulfoxide to increase movement of the protein past cell junctions (Fraser et al., 1987; Steele et al., 1996; Zhang and Sarras,
1994).
TGF-␤ SIGNALING: IT’S THERE BUT WE
DON’T YET KNOW WHAT IT DOES
Members of the TGF-␤ superfamily play prominent developmental signaling roles in bilaterians (Massague, 1998).
Genes for a type I TGF-␤ family receptor and two Smad
family transcription factors have been identified in the
coral Acropora (Samuel et al., 2001) and a gene encoding a
member of the bone morphogenetic (BMP) protein family
has been cloned from a sea anenome (Lelong et al., 2001).
Expression data for these genes have not been reported.
Recently, genes for two members of the BMP family have
been identified in Hydra and are in the process of being
characterized (B. Reinhardt and H. Bode, personal communication). cDNA clones encoding a chordin homologue (T.
Holstein, personal communication) and a R-Smad transcription factor (Hobmayer et al., 2001) have also been
isolated from Hydra. The R-Smad gene is expressed
throughout the endodermal epithelium and in the ectodermal epithelium of the body column. In addition, the
R-Smad gene is expressed in the nematocyte differentiation
pathway, and its expression is up-regulated in the oocyte
precursor cells of polyps undergoing oogenesis. Expression
of the chordin gene is restricted to the endodermal epithelium of the hypostome and the tentacles in the adult polyp
(T. Holstein, personal communication). During budding,
chordin expression begins in a small group of cells in the
endodermal epithelium at the stage when evagination has
begun. Expression is maintained throughout the bud
endoderm until the head structures are formed, at which
point expression becomes restricted to the head (T. Holstein, personal communication).
The data available to date do not allow one to develop a
clear hypothesis about how the BMP signaling pathway
works in Hydra. Knowledge of the expression pattern of the
receptors for the Hydra BMP homologues should help in
this regard. The expression information on chordin and
R-Smad suggests that this pathway plays multiple developmental roles in the polyp.
WHAT ABOUT NOTCH?
Developmental processes in bilaterians make heavy use
of the Notch signaling pathway (Mumm and Kopan, 2000).
Despite efforts by several labs, genes encoding Notch family members have not yet been identified in cnidarians.
Recently, however, genes encoding Hydra homologues of
Drosophila Suppressor of Hairless (a transcription factor in
the Notch pathway), hairy/Enhancer of Split (a target gene
in the Notch pathway), and Numb (an intracellular antagonist of Notch signaling) have been identified (P. Towb and
J. Posakony, personal communication). Thus, it seems
likely that the Hydra Notch pathway will be revealed in its
entirely in the not too distant future.
HEDGEHOG: NEMATODES DON’T NEED
IT, BUT HYDRA APPARENTLY DOES
The fourth member of the “big four” of developmental
signaling pathways (the other three being the Wnt, TGB-␤,
and Notch pathways) is the hedgehog pathway (Hammerschmidt et al., 1997). Until recently it was unclear whether
the hedgehog pathway is present in Hydra. Hedgehog itself
is absent from C. elegans, as is a gene for Smoothened, one
of the components of the hedgehog receptor (Ruvkun and
Hobert, 1998). Genes encoding other components of the
pathway, Patched (Kuwabara et al., 2000), another component of the hedgehog receptor, and Gli, a zinc finger
transcription factor (Knight and Shimeld, 2001), are present
in C. elegans. However, the complete hedgehog pathway is
present in Drosophila, another protostome, indicating that
portions of the hedgehog pathway have been secondarily
lost in C. elegans. Work recently carried out in the Galliot
lab (Kaloulis, 2000) has demonstrated that the hedgehog
pathway is indeed present in Hydra. So far, genes encoding
hedgehog itself and a member of the Gli/Ci family of
transcription factors have been isolated (Kaloulis, 2000).
The expression patterns of these genes will be of considerable interest.
© 2002 Elsevier Science (USA). All rights reserved.
209
Developmental Signaling in Hydra
HYDRA ON STEROIDS?
The nuclear receptor superfamily includes an array of
ligand-regulated transcription factors whose members include steroid receptors, retinoid receptors, and a large
number of so-called orphan receptors (Owen and Zelent,
2000). Members of the nuclear receptor superfamily, particularly the retinoid receptors, play crucial developmental
signaling roles in animals (Maden, 2000). Efforts to trace the
evolutionary history of the nuclear receptor superfamily in
animals have so far led to the identification of a single
member of this family in Hydra (Escriva et al., 1997).
Phylogenetic analysis of this Hydra receptor suggests that it
is most closely related to the COUP family within the
nuclear receptor superfamily. At the moment, we know
nothing more than this receptor. The roles of members of
the COUP family in other animals have been explored and
found to include developmental processes in a variety of
tissues, including the nervous system and the circulatory
system (Cooney et al., 2001).
The effects of exposure of adult Hydra polyps and aggregates of Hydra cells (which will regenerate polyps) to
various retinoids have been examined (Johnson and Chun,
1989). Concentrations were identified which were toxic to
both the adult polyp and the cell aggregates, but no specific
developmental effects were observed. In the absence of
information regarding the presence of retinoid receptors in
Hydra, it is not yet possible to determine the significance of
these results. It is interesting to note, however, that treatment of another hydrozoan, Hydractinia, with retinoids
results in patterning alterations (Müller, 1984).
RECEPTOR PROTEIN-TYROSINE KINASE
(RTK) PATHWAYS ARE THERE TOO
Given their importance in developmental signaling in
more recently diverged animals (Pawson and Bernstein,
1990; Shilo, 1992; Sternberg et al., 1995), RTKs and their
ligands are prime candidates for regulating developmental
fates in Hydra. Several RTK genes have been identified in
Hydra, some of which encode homologues of RTKs known
from bilaterians and others of which appear to be novel.
One of the Hydra RTK genes, termed HTK7, encodes a
member of the insulin receptor family (Steele et al., 1996).
The highest levels of expression of the HTK7 gene are in
nondividing ectodermal epithelial cells in the bases of the
tentacles and just above the foot basal disk (Steele et al.,
1996). A lower level of expression is seen in the epithelial
cells of the body column, and expression is absent from the
basal disk and the distal parts of the tentacles.
What could be the significance of the high level of
expression of the HTK7 gene in the proximal region of the
tentacles and just above the basal disk? The developmental
dynamics of Hydra and the results of studies of receptor
protein-tyrosine kinase signaling mechanisms in vertebrate
cells suggest a possibility. As discussed earlier, the tissue
dynamics of Hydra result in the continual displacement of
cells toward the extremities of the polyp where they are
then lost by sloughing from the tips of the tentacles and
from the basal disk (Fig. 1). Thus, epithelial cells in the body
column eventually move into the tentacles or the basal disk
where the ectodermal epithelial cells differentiate into
nondividing cell types (Fig. 2). We have hypothesized (Steele
et al., 1996) that a low level of the HTK7 receptor in the
body column results in the transduction of a signal that
stimulates cells to divide and that a high level of receptor
transduces a signal that stimulates cells to differentiate. In
the case of the tentacles, the differentiation event would be
conversion of body column epithelial cells into tentacle
battery cells. At the other end of the polyp, the differentiation event would be conversion of body column epithelial
cells into basal disk cells. This hypothesis is based on
findings from studies in mammalian systems showing that
RTK levels can determine whether a cell differentiates or
divides (Lillien, 1995; Marshall, 1995).
The lower level of HTK7 expression in the body column
is proposed to be sufficient to stimulate cell division but
not to trigger cell cycle arrest and differentiation into foot
or tentacle cells. Our finding that treatment of Hydra with
bovine insulin stimulates cell division of both ectodermal
and endodermal epithelial cells supports this portion of the
model (Steele et al., 1996). If our hypothesis is correct, why
does exogenously added insulin affect cell division but not
differentiation? One would argue that the level of receptor
in the body column is such that even extra insulin supplied
exogenously can do no more than increase the amount of
division since the level of receptor is too low to allow
sufficient signal to cause body column cells to differentiate
into basal disk or tentacle battery cells. Furthermore, in the
tentacles and the foot, the receptor would act simply as a
trigger for the differentiation events but would not provide
the specificity. This conclusion arises logically from the
fact that the receptor is present at high levels at both ends
of the animal, yet the two extremities produce very different types of cells. Thus, the specificity must be supplied by
other means. Interestingly, two homeobox genes are expressed in a pattern related to that of HTK7. One of the
genes, termed manacle, is expressed in the ring of cells at
the peduncle/basal disk boundary (Bridge et al., 2000). The
other gene, termed HyAlx, is expressed in a ring at the base
of the tentacle (Smith et al., 2000). These two genes may be
involved in mediating the response of cells to the signaling
cascade activated by HTK7.
Two other RTK genes with possible developmental roles
in Hydra are shin guard, which is expressed in a graded
pattern in the ectodermal epithelial cells of the pednucle
(Bridge et al., 2000), and Lemon, which is expressed in the
interstitial cell lineage and is up-regulated during gametogenesis and budding (Miller and Steele, 2000). Shin guard
has no homologue in other animals. Its extracellular domain contains EGF repeats and its closest relative is another Hydra RTK (Reidling et al., 2000) which appears not
to have a developmental role. Shin guard expression ap-
© 2002 Elsevier Science (USA). All rights reserved.
210
Robert Steele
pears to be controlled by a signal from the foot (Bridge et al.,
2000). Its graded expression pattern (high near the basal disk
and low near the budding zone) is intriguing since other
studies have shown that the tissue of the peduncle apparently becomes committed to foot formation as it is displaced toward the basal end of the polyp (Bode and Bode,
1984a). Shin guard might play a role in this commitment
process.
The Hydra Lemon RTK (Miller and Steele, 2000) is
homologous to the human CCK-4, the chicken Klg, and the
Drosophila Dtrk RTKs (Kroiher et al., 2001). These three
RTKs are unusual in that they lack kinase activity due to
amino acid substitutions in the catalytic domain. This lack
of activity makes their role in the cell puzzling. Lemon’s
expression pattern during budding and gametogenesis suggests the possibility that it plays a role in altering the cell
cycle kinetics in the interstitial cell compartment during
these developmental processes (Miller and Steele, 2000).
DOES HYDRA USE ENDOTHELIN AS
A DEVELOPMENTAL SIGNALING
MOLECULE?
In efforts to identify metalloproteinases which act on the
extracellular matrix of Hydra, the Sarras lab has identified
a possible homologue of the vertebrate endothelin converting enzyme (ECE) (Zhang et al., 2001). In vertebrates, ECE
catalyzes the final proteolytic step in the production of
mature endothelins (Rubanyi and Polokoff, 1994). Although
their most well-studied role is the regulation of vascular
tone, endothelins are apparently also involved in craniofacial development (Clouthier et al., 1998; Kurihara et al.,
1994; Yanagisawa et al., 1998).
Using antibodies against human endothelin-1, Zhang et
al. (2001) were able to detect immunoreactive material in
Hydra lysates. Furthermore, the immunoreactive material
was enriched in the foot fraction. Upon treatment with
human endothelin-1 or endothelin-3, Hydra polyps contract to about half their normal length within 2.5 h. No
contraction was seen upon treatment with endothelin-2. In
situ hybridization with a probe for the Hydra ECE gene
showed that the gene is expressed throughout the polyp
(Zhang et al., 2001). The highest levels of expression are
seen in the endodermal epithelial cells at the bases of the
tentacles and in the enodermal epithelial cells in the foot.
Expression of the ECE gene in the foot endoderm is a
relatively early event during foot regeneration, starting at
3 h after removal of the foot.
To test whether ECE is required for foot formation,
polyps were treated with antisense phosphorothioate oligonucleotides directed against various portions of the ECE
gene. The oligonucleotides were applied by localized electroporation into the endodermal epithelium at the basal
pole following foot removal. Three of the antisense oligonucleotides caused a delay in foot regeneration in a majority
of the treated animals (Zhang et al., 2001).
Taken together, the data of Zhang et al. (2001) suggest
that a pathway involving an endothelin-related molecule is
involved in both maintaining the state of contraction in the
polyp and in the formation of the foot. Identification of the
presumed endothelin-related molecule and its receptor are
the obvious next steps in defining how this pathway functions in Hydra.
SIGNALING VIA THE EXTRACELLULAR
MATRIX
Although originally not thought of as a signaling entity,
the extracellular matrix is now recognized to play an
important role in conveying signals to cells (De Arcangelis
and Georges-Labouesse, 2000). Such signaling is achieved
by interaction of the matrix molecules with receptors of the
integrin family. While a Hydra integrin has not yet been
characterized, cDNAs for integrins from two other cnidarians (the coral Acropora and the colonial marine hydrozoan
Podocoryne) have been reported (Brower et al., 1997; ReberMuller et al., 2001). The genes for several extracellular
matrix molecules have been cloned from Hydra and the
molecules have been shown to be involved in morphogenesis and cell division (Sarras and Deutzmann, 2001). Identification of Hydra integrins and the molecules which act
downstream of them will provide the tools necessary for
investigating this important signaling pathway in greater
detail.
A ROLE FOR SRC IN HEAD FORMATION:
THE USE OF SPECIFIC INHIBITORS
Molecules which are known to inhibit or activate signaling pathways in other systems, such as cultured mammalian cells, are potentially powerful tools for dissecting the
roles of signaling pathways in Hydra. What is required is
that the target protein be present in Hydra and that it be
well enough conserved to be affected by the molecule. This
is likely to be the situation in a number of cases, but so far
we don’t know how often. Proof of principle for this
approach has been provided by a study from Cardenas et al.
(2000) using PP1, an inhibitor specific for Src family
protein-tyrosine kinases (PTKs) in vertebrates (Hanke et al.,
1996). Hydra has only a single Src family PTK, whose gene
has been cloned (Bosch et al., 1989). Hydra Src is inhibited
by PP1 in an in vitro kinase assay (L. Salgado, personal
communication). To test the effect of PP1 on Hydra development, Cardenas et al. (2000) cut off both the head and the
foot of adult Hydra polyps and followed the course of
regeneration in the presence or absence of inhibitor. At a
PP1 concentration of 1 ␮M, head regeneration was delayed
by 3 days relative to control animals, while foot regeneration was only slightly delayed. The lack of an effect on foot
regeneration is not due to the absence of Src in the lower
portion of the animal; the Src gene is expressed throughout
© 2002 Elsevier Science (USA). All rights reserved.
211
Developmental Signaling in Hydra
the polyp (Miller et al., 2000). The delaying effect was not
seen if PP1 exposure began at 10 h following removal of the
head, indicating that the inhibitor-sensitive step occurs
early in the regeneration process.
To test whether the effect of PP1 on head regeneration
was due to inhibition of cell division, Cardenas et al. (2000)
examined the labeling index of cells in epithelial Hydra
treated with PP1. The polyps were decapitated, incubated
for 24 – 48 h in the presence or absence of PP1, pulsed for 4 h
with bromodeoxyuridine, and the labeled cells were then
counted. No difference was seen between the treated and
control animals.
The minimal effect of PP1 on foot regeneration in polyps
from which the head and foot have been removed is perhaps
surprising in light of data indicating that foot formation
depends on the presence of a head (Müller, 1990). This
discrepancy can be explained if PP1 blocks at a point after
the foot “assistance” process is initiated but prior to the
completion of head regeneration.
By examining PP1-treated animals for expression of various genes associated with head formation, it should be
possible to determine at what point the block to regeneration occurs and therefore at what point Src is required in the
regeneration process.
ONE OF HYDRA’S RAS GENES HAS A
ROLE IN HEAD FORMATION
Ras is a key component in signal transduction pathways
in animal cells. Its role has been particularly well worked
out in pathways involving RTKs (Tan and Kim, 1999).
While Ras activation has not yet been functionally linked
to any RTKs in Hydra, the expression pattern of one of the
Ras genes in Hydra indicates that it plays a role in head
formation. Hydra contains at least two Ras genes (Ras1 and
Ras2), both of which appear to be expressed througout the
polyp (Bosch et al., 1995). The potential involvement of the
Ras genes in head formation has been examined by following expression during head regeneration. Ras1 and Ras2 are
normally expressed at comparable levels in the head and the
body. Removal of the head leads to a dramatic drop in the
level of Ras2 RNA in the upper portion of the body column,
while expression in the lower portion of the body column is
unaffected (Bosch et al., 1995). The level of Ras1 RNA does
not change in either the upper or lower part of the body
column of animals from which the head has been removed.
By 8 h following decapitation, the Ras2 RNA level has
returned to normal in the upper body column.
Treatment of adult Hydra polyps with the phorbol ester
12-O-tetradecanoylphorbol-13-acetate (TPA) results in
many of the treated polyps forming ectopic heads on the
body column (Müller, 1990). Treatment of decapitated
polyps for 20 min with TPA blocked the decrease in Ras2
RNA in the upper body column seen in untreated animals
(Bosch et al., 1995). These results were interpreted as
indicating that a signal from the head, acting through
protein kinase C, is required to maintain the level of Ras2
RNA normally found in the head. These findings raise a
number of interesting questions. If the level of Ras2 RNA is
normally the same throughout the animal, what purpose
would the transient drop in the upper body column during
head regeneration serve? What establishes the boundary
between the region where RNA decreases and the region
where it doesn’t? Does the level of Ras2 protein drop as a
result of the drop in Ras2 RNA? If so, how is the turnover of
the protein controlled? Hopefully, it will eventually be
possible to address these questions.
A final question is whether a change in the level of Ras
protein can affect the outcome of a signaling process in
which Ras is involved. That the level of Ras activity can
effect the outcome of a developmental process has been
nicely demonstrated in Drosophila (Greenwood and Struhl,
1997). However, it is not clear whether the level of Ras
activity available to a pathway is necessarily directly proportional to the amount of Ras protein. Expression of high
levels of normal Ras protein under control of the hsp70
promoter had no effect on Drosophila development (Bishop
and Corces, 1988). In contrast to this result, increasing the
gene dosage of normal Ras in C. elegans leads to a multivulval phenotype, consistent with the interpretation that
increasing the level of Ras protein leads to an increase in
the level of Ras activity (Sternberg and Han, 1998). Thus,
the changes in Ras2 expression seen during head regeneration in Hydra may very well lead to changes in the output
from one or more signaling pathways.
G PROTEIN-COUPLED SEVENTRANSMEMBRANE RECEPTORS: WILL
THEY BE FOUND TO PLAY AN
IMPORTANT ROLE IN HYDRA
DEVELOPMENT?
The G protein-coupled seven-transmembrane receptors
(GPCRs) are an ancient family predating the appearance of
the first metazoans, and they are used in a variety of
settings in animals. Thus, it is perhaps surprising that so far
none of this class of receptor has been identified in Hydra.
In fact, only two members of the family have been reported
in the phylum Cnidaria, one in a sea anemone (Nothacker
and Grimmelikhuijzen, 1993) and one in Hydractinia (GenBank Accession No. AJ440242). Many of the neuropeptides
in vertebrates act through receptors of this class, so it is
hard to avoid the idea that at least some of the peptides in
Hydra would use such receptors. This is, however, an
untested notion. The apparent signaling by head activator
through an LDL receptor relative gives one pause in assuming that GPCRs will play a major role in developmental
signaling pathways in Hydra. Efforts to identify the receptors for the peptides produced by the Hydra Peptide Project
and EST sequencing projects should reveal how much
Hydra developmental signaling depends GPCR’s.
© 2002 Elsevier Science (USA). All rights reserved.
212
Robert Steele
GETTING HYDRA TO MAKE EXTRA
HEADS AND EXTRA FEET: IT’S EASIER
THAN IT MIGHT SEEM
Gene cloning efforts have revealed the presence of a
number of Hydra homologues of developmental signaling
molecules known from bilaterians. Ironically, however,
some of the most dramatic developmental effects associated with manipulation of signaling pathways in Hydra
have been obtained not by cloning genes and attempting to
alter their expression but rather by simply incubating the
animals in the presence of compounds such as diacylglycerol (DAG) (Müller, 1989, 1990) or lithium chloride (Hassel
et al., 1993; Hassel and Berking, 1990). Daily exposure to
DAG leads, in about a week, to the appearance of clusters of
ectopic tentacles on the body column (Müller, 1989). Subsequently, hypostomes form in the tentacle clusters and
secondary polyps arise from the treated animal. These
results have been interpreted as being due to activation of
protein kinase C, a reasonable hypothesis, but data that
prove this have not yet been obtained. Two PKC genes have
been identified in Hydra (Hassel, 1998; Hassel et al., 1998).
However, the proteins encoded by these genes have not yet
been produced in heterologous systems or purified from
Hydra so that their response to DAG can be measured in
direct biochemical tests. Hydra extracts do contain kinase
activity that is stimulated by calcium plus diacylglycerol
and phosphatidylserine (Hassel et al., 1998), as expected for
PKC.
Treatment of Hydra with lithium can lead to one of two
results, depending on the treatment protocol that is used.
Continuous exposure to a low lithium concentration (0.5–1
mM) over several days leads to the development of ectopic
feet on the body column (Hassel and Berking, 1990). Interestingly, this response to lithium is seen in some species of
Hydra (H. vulgaris) but not in others (H. magnipapillata)
(Hassel and Berking, 1990). While continuous exposure to a
low concentration of lithium leads to ectopic foot formation, a short exposure (2 days) to a high concentration of
lithium, (4 mM) followed by continuous exposure to 1 mM
lithium, leads first to formation of ectopic heads and
tentacles in the upper body column, followed by formation
of ectopic feet in the lower body column (Hassel et al.,
1993). The dramatic effects of lithium on Hydra patterning
are not easy to interpret mechanistically. The processes
underlying lithium’s effects on Hydra patterning are likely
to be complicated since lithium is potentially targeting
more than one molecule in Hydra cells. Possible targets
include GSK-3 and inositol monophosphatase (Phiel and
Klein, 2001).
Both DAG and lithium have been used extensively in
combination with analyses of gene expression to dissect
developmental processes in Hydra. By determining how the
expression pattern of a developmentally important gene is
changed in DAG- or lithium-treated polyps, one can gain
insight into how signaling pathways may interact with the
expression of such genes. Examples of results from such
experiments are the findings that lithium treatment shifts
the apical expression boundaries of genes expressed in the
basal part of the animal more toward the head (Bridge et al.,
2000; Grens et al., 1996), in keeping with the findings that
continuous exposure to a low concentration of lithium
drives tissue toward a basal positional value (Hassel et al.,
1993; Hassel and Berking, 1990).
While DAG and lithium are valuable tools for analyzing
developmental processes in Hydra, we don’t yet have any
idea about what signaling pathways they perturb. The Wnt
pathway is one possible target due to lithium’s inhibitory
effects on GSK-3. The phosphoinositide cycle is likely to be
a component of more than one signaling pathway in Hydra
and thus it may be difficult to determine exactly what
signals are upstream of effects that DAG and/or lithium
have on this cycle.
SO WHAT DO WE KNOW?
From the data currently in hand, we can begin to see the
outline of what kinds of signaling pathways it takes to build
a Hydra. One thing that is abundantly clear is that nearly
all of the developmental pathways that are so familiar from
studies of model bilaterians were present in the last common ancestor of these organisms and Hydra. Thus, the
basic machinery for making a metazoan was invented very
early and much of the diversity we see in animal morphology must have been the result of working with this machinery
The thing that is very surprising is the abundance of
peptide signaling molecules in Hydra. We are just beginning to understand what these molecules can do, so it is at
present difficult to place them in an evolutionary perspective. It will be very important to try to identify genes
encoding homologues of these peptides in other organisms.
This will require search algorithms designed to identify
short coding homologies with such motifs as proteolytic
processing signals (e.g., dibasic cleavage sites). A gene
encoding members of the LWamide peptide family, one of
the peptide families identified by the Hydra Peptide Project
(Bosch and Fujisawa, 2001; Takahashi et al., 1997), has
recently been identified in C. elegans (T. Fujisawa, personal
communication). The gene is expressed in the AIA interneurons, which are in the nerve ring encircling the pharynx.
Mutants in the C. elegans LWamide gene have been generated, and the resulting phenotype is in the process of being
characterized (T. Fujisawa, personal communication).
If many of the Hydra peptides are used in other organisms
(particularly those with tractable genetics), it will be very
interesting to see what they do there. If, on the other hand,
other organisms are found to lack most of the peptides
present in Hydra, we will be witness to a remarkable
experiment in evolution that will have much to say about
how multicellular animals operate.
© 2002 Elsevier Science (USA). All rights reserved.
213
Developmental Signaling in Hydra
WHAT CAN WE DO (OR HOPE TO DO)
WITH HYDRA TO DISSECT SIGNALING
PATHWAYS?
On the basis of the large number of question marks in
Table 1, it is obvious that we have a number of promising
leads but that we have barely scratched the surface with
regard to understanding the signaling pathways which regulate developmental processes in Hydra. How should we
proceed if we want to fully understand these pathways? A
review of various approaches that might be taken is given
below.
Genetic Screens
Efforts were initiated by Sugiyama (1983) some years ago
to apply genetic methods to understanding developmental
processes in Hydra. By crossing of animals from the same
pond (to increase the chances of producing progeny which
are homozygous for recessive alleles due to inbreeding),
Sugiyama identified a number of interesting mutant strains.
These included, among others, a mutant in which head
regeneration is inhibited (Sugiyama and Fujisawa, 1977) and
mutants with altered body sizes (Takano and Sugiyama,
1985). Some of these mutants are being used in molecular
studies, and they can provide valuable information (Endl et
al., 1999; Technau and Bode, 1999). However, in no case do
we know the site of the mutation and there is little hope of
obtaining this information in the foreseeable future. At
present, no genetic screens are underway using Hydra. Such
screens face formidable roadblocks. It would be very difficult to obtain Hydra embryos in the numbers necessary to
carry out such screens, and the hatching of the embryos
occurs over a period of weeks to months following a period
of apparent diapause. This is not to say, however, that all
cnidarians are intractable as genetic systems. The colonial
marine hydrozoan Hydractinia has been used successfully
in a genetic screen for the loci which control allorecognition in this genus (Mokady and Buss, 1996). The sea
anemone Nematostella vectensis also shows promise as a
genetic screening system since it produces large numbers of
embryos which complete development from fertilized eggs
to juvenile polyps in 1 week (Hand and Uhlinger, 1992).
Sexual maturity in Nematostella is reached in about 4
months. Thus, it seems probable that genetic screens for
developmental regulatory genes in a cnidarian will eventually be carried out using Nematostella. Genes discovered in
such screens would obviously be valuable tools for further
studies in Hydra.
The Prospects for Transgenic Hydra
While classical genetic tools are not readily applicable to
Hydra, one can envision a number of informative experiments that could be carried out if transgenic methods could
be brought to bear on this organism. Attempts to introduce
cloned genes into Hydra have been carried out sporadically
by a number of researchers over the past 15 years. A
promising initial result showed that DNA could be introduced into the cells of adult polyps by electroporation (T.
Bosch, A. Grens, H. Bode, and R. Steele, unpublished
observations) and that low level expression of exogenous
genes could be obtained. However, the introduced DNA
was not retained in the polyp, being lost after a few days.
Recently, a GFP gene under the control of a Hydra actin
gene promoter has been shown to be expressed following
electroporation into adult polyps (C. David, personal communication). While expression is lost after a few days, as
was seen in earlier studies, the use of GFP as a reporter will
greatly facilitate attempts to improve delivery and retention of the transgene. Other possible delivery methods
besides electroporation include the use of pantropic retroviruses (Yee et al., 1994) and transposable elements (Robertson, 1997).
Blockage of Gene Expression Using Antisense
Oligonucleotides
Applications of antisense oligonucleotides as inhibitors
of gene expression have been carried out on a variety of
developmental systems over a number of years. In some
cases, the results have been convincing, while in other
cases, it has been difficult to rule out the nonspecific effects
that can be associated with this technology. Carefully
controlled experiments using phosphorthioate antisense
oligonucleotides in Hydra have yielded informative results
(Deutzmann et al., 2000; Fowler et al., 2000; Zhang et al.,
2001). Morpholino antisense oligonucleotides (Corey and
Abrams, 2001; Ekker, 2000; Nasevicius and Ekker, 2000)
have not yet been successfully tested in Hydra, but their
increased specificity relative to phosphorthioate oligonucleotides should make them useful tools for studies in
Hydra.
RNA Interference
One of the more remarkable biological phenomena identified in recent years is RNA interference, a process in
which double-stranded RNA produced from a cloned segment of a particular gene is capable of specifically inhibiting the expression of the gene from which it was derived
(Bosher and Labouesse, 2000). This technology has been
most successfully applied in the nematode C. elegans, but
the phenomenon has now been reported in a number of
other animals, including Hydra. Initial results showed that
introduction of dsRNA from a gene expressed only in the
head (the ks1 gene) into Hydra by electroporation resulted
in a lowering of the level of the endogenous target mRNA
and a slowing of the process of head regeneration (Lohmann
et al., 1999). Subsequently RNAi has been used successfully
© 2002 Elsevier Science (USA). All rights reserved.
214
Robert Steele
with two additional Hydra genes, the HEADY peptide gene
(see above) and the HyA1x homeobox gene (Smith et al.,
2000). Localized electroporation of HyAlx dsRNA into early
stage buds delayed the appearance of tentacles on the buds,
a result in keeping with the apparent role of HyA1x in
tentacle formation.
While RNAi works in Hydra, the effects can be relatively
modest. Therefore, it will be useful to attempt to refine the
technique based on biochemical parameters using extracts
as has been done in other systems (Tuschl et al., 1999).
Secondly, epistasis experiments are needed to show that the
blocks achieved by RNAi are located in different positions.
This will not be an easy undertaking given the relatively
short time frames involved and the fact that in situ hybridization will be needed in most cases to follow gene expression. Nonetheless, such studies are critical if we hope to use
RNAi to dissect developmental pathways in Hydra.
Matching Up Ligands and Receptors
The Hydra Peptide Project and the ease of isolating the
genes for some classes of receptors (e.g., RTKs) using PCR
has given us a wealth of ligands for which we as yet have no
receptors and receptors for which we have no ligands.
Various approaches are available for identifying both orphan receptors and orphan ligands (Cheng and Flanagan,
2001; Civelli et al., 2001) and these should be applicable to
Hydra.
Genomics
A great deal of molecular biological slogging to understand developmental signaling in Hydra could be avoided if
we had the sequence of the Hydra genome in hand. One
could then potentially identify the Hydra homologues of
any developmental signaling molecules known from other
animals. In addition, it would be possible to identify members of signaling molecule families which may not have
homologues in other animals (e.g. Hydra-specific RTKs and
seven-transmembrane receptors). While there is no effort
currently underway to sequence the Hydra genome, the
National Science Foundation has funded a project which
will result in the production of approximately 50,000 expressed sequence tags (EST’s) from Hydra. Such sequences
will undoubtedly prove to be a gold mine for the Hydra
research community as well as for other investigators
interested in the evolution of metazoans.
Screening with Combinatorial Chemical Libraries
The fact that Hydra polyps are essentially naked epithelia
which live in a simple, defined culture medium would seem
to make them a promising system for screening for developmental effects with combinatorial libraries of chemical
compounds. This approach has been applied with success to
zebrafish embryos (Peterson et al., 2000). One could, for
example, imagine screening with Hydra for compounds
which give neomorphic phenotypes (e.g., extra heads or
feet) or which inhibit developmental processes (e.g., budding or regeneration). Diacylglycerol and PP1 are examples
of chemical compounds of the sort which one would hope
to identify with a combinatorial library screen. The ability
of individual Hydra polyps to be cultured in 96-well plates
offers the potential for such a screen to be carried out in a
high throughput mode. The drawback of a combinatorial
library screen is the difficulty of identifying the target
molecules of the active compounds.
COMBINING THE OLD AND THE NEW
I will finish this review with a reminder of what enticing
experimental possibilities await us when we are able to
combine the powerful tools with which molecular biology
is currently practiced with the equally powerful methods
that Hydra researchers have had at their disposable in some
cases since Trembley’s studies of more than 200 years ago
(Trembley, 1744). One can imagine a situation where grafting experiments will involve using pieces of tissue from a
transgenic donor Hydra producing GFP and a nontransgenic
host. It will then be possible to follow individual GFPproducing cells with great precision as the cells induce a
secondary axis, are displaced through the host polyp into
the foot or head, or become incorporated into a bud. Or
imagine what one might see if one made a chimera in which
the interstitial cell lineage produced GFP but the epithelial
cells didn’t. Or let’s get really crazy and imagine an animal
in which a portion of the body column contains an hsp70
promoter-driven transgene for Wnt but the rest of the polyp
doesn’t; producing such a graft would be simple if the
transgenic animals were available. What would happen
when such an animal is heat-shocked? Such an experiment
probably isn’t as far away as it may seem. A variety of genes
of interest have already been cloned and more are on the
way. And the Hydra hsp70 promoter is in hand (Gellner et
al., 1992).
ACKNOWLEDGMENTS
Work in my laboratory has been carried out with generous
support from the National Science Foundation. I am grateful to the
members of the Hydra research community for generously communicating their unpublished results. This paper is dedicated to
my brother David, for continually reminding me that there is a
world outside the ivory tower of academe.
Note added in proof. A paper demonstrating expression of GFP
transgenes has recently been published by Miljkovic et al. (Dev.
Biol. 246, 377–390, 2002).
© 2002 Elsevier Science (USA). All rights reserved.
215
Developmental Signaling in Hydra
REFERENCES
Adoutte, A., Balavoine, G., Lartillot, N., Lespinet, O., Prud’homme,
B., and de Rosa, R. (2000). The new animal phylogeny: Reliability
and implications. Proc. Natl. Acad. Sci. USA 97, 4453– 4456.
Bishop, J. G., 3rd, and Corces, V. G. (1988). Expression of an
activated ras gene causes developmental abnormalities in transgenic Drosophila melanogaster. Genes Dev. 2, 567–577.
Bode, H. R. (1992). Continuous conversion of neuron phenotype in
hydra. Trends Genet. 8, 279 –284.
Bode, H. R. (1996). The interstitial cell lineage of hydra: A stem cell
system that arose early in evolution. J. Cell Sci. 109, 1155–1164.
Bode, H. R., Berking, S., David, C. N., Gierer, A., Schaller, C. H.,
and Trenkner, E. (1973). Quantitative analysis of cell types
during growth and morphogenesis in hydra. Roux’s Arch. Dev.
Biol. 171, 269 –285.
Bode, P. M., and Bode, H. R. (1984a). Formation of pattern in
regenerating tissue pieces of Hydra attenuata. II. Degree of
proportion regulation is less in the hypostome and tentacle zone
than in the tentacles and basal disc. Dev. Biol. 103, 304 –312.
Bode, P. M., and Bode, H. R. (1984b). Patterning in Hydra: In
“Pattern Formation, A Primer in Developmental Biology” (G. M.
Malacinski and S. V. Bryant, Eds.), pp. 213–241. Macmillan, New
York.
Bodenmuller, H., Schilling, E., Zachmann, B., and Schaller, H. C.
(1986). The neuropeptide head activator loses its biological
activity by dimerization. EMBO J. 5, 1825–1829.
Bosch, T. C., Benitez, E., Gellner, K., Praetzel, G., and Salgado,
L. M. (1995). Cloning of a ras-related gene from Hydra which
responds to head-specific signals. Gene 167, 191–195.
Bosch, T. C., and Fujisawa, T. (2001). Polyps, peptides and patterning. Bioessays 23, 420 – 427.
Bosch, T. C., and Lohmann, J. U. (1998). Identification of differentially expressed genes by nonradioactive differential display of
messenger RNA. Methods Mol. Biol. 86, 153–160.
Bosch, T. C. G., Unger, T. F., Fisher, D. A., and Steele, R. E. (1989).
Structure and expression of STK, a src-related gene in the simple
metazoan Hydra attenuata. Mol. Cell. Biol. 9, 4141– 4151.
Bosher, J. M., and Labouesse, M. (2000). RNA interference: Genetic
wand and genetic watchdog. Nat. Cell Biol. 2, E31–E36.
Bridge, D. M., Stover, N. A., and Steele, R. E. (2000). Expression of
a novel receptor tyrosine kinase gene and a paired-like homeobox
gene provides evidence of differences in patterning at the oral and
aboral ends of hydra. Dev. Biol. 220, 253–262.
Broun, M., and Bode, H. R. (2002). Characterization of the head
organizer in hydra. Development 129, 875– 884.
Brower, D. L., Brower, S. M., Hayward, D. C., and Ball, E. E. (1997).
Molecular evolution of integrins: Genes encoding integrin beta
subunits from a coral and a sponge. Proc. Natl. Acad. Sci. USA
94, 9182–9187.
Campbell, R. D. (1967a). Tissue dynamics of steady state growth in
Hydra littoralis. I. Patterns of cell division. Dev. Biol. 15,
487–502.
Campbell, R. D. (1967b). Tissue dynamics of steady-state growth in
Hydra littoralis. II. Patterns of tissue movement. J. Morphol. 121,
19 –28.
Campbell, R. D. (1973). Vital marking of single cells in developing
tissues: India ink injection to trace tissue movements in hydra.
J. Cell Sci. 13, 651– 661.
Campbell, R. D. (1976). Elimination by Hydra interstitial and nerve
cells by means of colchicine. J. Cell Sci. 21, 1–13.
Campbell, R. D., and David, C. N. (1974). Cell cycle kinetics and
development of Hydra attenuata. II. Interstitial cells. J. Cell Sci.
16, 349 –358.
Cardenas, M., Fabila, Y. V., Yum, S., Cerbon, J., Bohmer, F. D.,
Wetzker, R., Fujisawa, T., Bosch, T. C., and Salgado, L. M. (2000).
Selective protein kinase inhibitors block head-specific differentiation in hydra. Cell Signal. 12, 649 – 658.
Cavalier-Smith, T., Allsopp, M. T. E. P., Chao, E. E., Boury-Esnault,
N., and Vacelet, J. (1996). Sponge phylogeny, animal monophyly,
and the origin of the nervous system: 18S rRNA evidence. Can.
J. Zool. 74, 2031–2045.
Cheng, H. J., and Flanagan, J. G. (2001). Cloning and characterization of RTK ligands using receptor-alkaline phosphatase fusion
proteins. Methods Mol. Biol. 124, 313–334.
Civelli, O., Nothacker, H. P., Saito, Y., Wang, Z., Lin, S. H., and
Reinscheid, R. K. (2001). Novel neurotransmitters as natural
ligands of orphan G-protein-coupled receptors. Trends Neurosci.
24, 230 –237.
Clouthier, D. E., Hosoda, K., Richardson, J. A., Williams, S. C.,
Yanagisawa, H., Kuwaki, T., Kumada, M., Hammer, R. E., and
Yanagisawa, M. (1998). Cranial and cardiac neural crest defects in
endothelin-A receptor-deficient mice. Development 125, 813–
824.
Collins, A. G. (1998). Evaluating multiple alternative hypotheses
for the origin of bilateria: An analysis of 185 rRNA molecular
evidence. Proc. Natl. Acad. Sci. USA 95, 15458 –15463.
Collins, A. G. (2000). Towards understanding the phylogenetic
history of Hydrozoa: Hypothesis testing with 18S gene sequence
data. Scientia Marina 64 (suppl. 1), 5–22.
Cooney, A. J., Lee, C. T., Lin, S., Tsai, S. Y., and Tsai, M. (2001).
Physiological function of the orphans GCNF and COUP-TF.
Trends Endocrinol. Metab. 12, 247–251.
Corey, D. R., and Abrams, J. M. (2001). Morpholino antisense
oligonucleotides: Tools for investigating vertebrate development. Genome Biol. 2.
De Arcangelis, A., and Georges-Labouesse, E. (2000). Integrin and
ECM functions: Roles in vertebrate development. Trends Genet.
16, 389 –395.
Deutzmann, R., Fowler, S., Zhang, X., Boone, K., Dexter, S.,
Boot-Handford, R. P., Rachel, R., and Sarras, M. P., Jr. (2000).
Molecular, biochemical and functional analysis of a novel and
developmentally important fibrillar collagen (Hcol-I) in hydra.
Development 127, 4669 – 4680.
Dübel, S., Hoffmeister, S. A. H., and Schaller, H. C. (1987).
Differentiation pathways of ectodermal epithelial cells in hydra.
Differentiation 35, 181–189.
Ekker, S. C. (2000). Morphants: A new systematic vertebrate
functional genomics approach. Yeast 17, 302–306.
Endl, I., Lohmann, J. U., and Bosch, T. C. (1999). Head-specific gene
expression in Hydra: Complexity of DNA–protein interactions at
the promoter of ks1 is inversely correlated to the head activation
potential. Proc. Natl. Acad. Sci. USA 96, 1445–1450.
Escriva, H., Safi, R., Hanni, C., Langlois, M. C., Saumitou-Laprade,
P., Stehelin, D., Capron, A., Pierce, R., and Laudet, V. (1997).
Ligand binding was acquired during evolution of nuclear receptors. Proc. Natl. Acad. Sci. USA 94, 6803– 6808.
Fowler, S. J., Jose, S., Zhang, X., Deutzmann, R., Sarras, M. P., Jr.,
and Boot-Handford, R. P. (2000). Characterization of hydra type
IV collagen. Type IV collagen is essential for head regeneration
and its expression is up-regulated upon exposure to glucose.
J. Biol. Chem. 275, 39589 –39599.
© 2002 Elsevier Science (USA). All rights reserved.
216
Robert Steele
Franke, I., Buck, F., and Hampe, W. (1997). Purification of a
head-activator receptor from hydra. Eur. J. Biochem. 244, 940 –
945.
Fraser, S. E., Green, C. R., Bode, H. R., and Gilula, N. B. (1987).
Selective disruption of gap junctional communication interferes
with a patterning process in hydra. Science 237, 49 –55.
Galliot, B. (2000). Conserved and divergent genes in apex and axis
development of cnidarians. Curr. Opin. Genet. Dev. 10, 629 –
637.
Galliot, B., Welschof, M., Schuckert, O., Hoffmeister, S., and
Schaller, H. C. (1995). The cAMP response element binding
protein is involved in hydra regeneration. Development 121,
1205–1216.
Gellner, K., Praetzel, G., and Bosch, T. C. (1992). Cloning and
expression of a heat-inducible hsp70 gene in two species of Hydra
which differ in their stress response. Eur. J. Biochem. 210,
683– 691.
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. Nat. New Biol. 239, 98 –101.
Greenwood, S., and Struhl, G. (1997). Different levels of Ras
activity can specify distinct transcriptional and morphological
consequences in early Drosophila embryos. Development 124,
4879 – 4886.
Grens, A., Gee, L., Fisher, D. A., and Bode, H. R. (1996). CnNK-2, an
NK-2 homeobox gene, has a role in patterning the basal end of
the axis in hydra. Dev. Biol. 180, 473– 488.
Grens, A., Shimizu, H., Hoffmeister, S. A., Bode, H. R., and
Fujisawa, T. (1999). The novel signal peptides, pedibin and
Hym-346, lower positional value thereby enhancing foot formation in hydra. Development 126, 517–524.
Grimmelikhuijzen, C. J. P. (1979). Properties of the foot activator
from hydra. Cell Differ. 8, 267–273.
Grimmelikhuijzen, C. J. P., and Schaller, H. C. (1977). Isolation of
a substance activating foot formation in hydra. Cell Differ. 6,
297–305.
Hammerschmidt, M., Brook, A., and McMahon, A. P. (1997). The
world according to hedgehog. Trends Genet. 13, 14 –21.
Hampe, W., Urny, J., Franke, I., Hoffmeister-Ullerich, S. A., Herrmann, D., Petersen, C. M., Lohmann, J., and Schaller, H. C.
(1999). A head-activator binding protein is present in hydra in a
soluble and a membrane-anchored form. Development 126,
4077– 4086.
Hand, C., and Uhlinger, K. R. (1992). The culture, sexual and
asexual reproduction, and growth of the sea anemone Nematostella vectensis. Biol. Bull. 182, 169 –176.
Hanke, J. H., Gardner, J. P., Dow, R. L., Changelian, P. S., Brissette,
W. H., Weringer, E. J., Pollok, B. A., and Connelly, P. A. (1996).
Discovery of a novel, potent, and Src family-selective tyrosine
kinase inhibitor. Study of Lck- and FynT-dependent T cell
activation. J. Biol. Chem. 271, 695–701.
Harafuji, N., Takahashi, T., Hatta, M., Tezuka, H., Morishita, F.,
Matsushima, O., and Fujisawa, T. (2001). Enhancement of foot
formation in Hydra by a novel epitheliopeptide, Hym- 323.
Development 128, 437– 446.
Harrison, F. W., and Westfall, J. A. (1991). Placozoa, Porifera,
Cnidaria, and Ctenophora. In “Microscopic Anatomy of Invertebrates,” Vol. 2. Wiley-Liss, New York.
Hassel, M. (1998). Upregulation of a Hydra vulgaris cPKC gene is
tightly coupled to the differentiation of head structures. Dev.
Genes Evol. 207, 489 –501.
Hassel, M., Albert, K., and Hofheinz, S. (1993). Pattern formation in
Hydra vulgaris is controlled by lithium-sensitive processes. Dev.
Biol. 156, 362–371.
Hassel, M., and Berking, S. (1990). Lithium ions interfere with
pattern control in Hydra vulgaris. Roux’s Arch. Dev. Biol. 198,
382–388.
Hassel, M., Bridge, D. M., Stover, N. A., Kleinholz, H., and Steele,
R. E. (1998). The level of expression of a protein kinase C gene
may be an important component of the patterning process in
Hydra. Dev. Genes Evol. 207, 502–514.
Herz, J. (2001). The LDL receptor gene family: (Un)expected signal
transducers in the brain. Neuron 29, 571–581.
Hobmayer, B., Holstein, T. W., and David, C. N. (1997). Stimulation of tentacle and bud formation by the neuropeptide head
activator in Hydra magnipapillata. Dev. Biol. 183, 1– 8.
Hobmayer, B., Rentzsch, F., and Holstein, T. W. (2001). Identification and expression of HySmadl, a member of the R-Smad family
of TGFbeta signal transducers, in the diploblastic metazoan
Hydra. Dev. Genes Evol. 211, 597– 602.
Hobmayer, B., Rentzsch, F., Kuhn, K., Happel, C. M., von Laue,
C. C., Snyder, P., Rothbacher, U., and Holstein, T. W. (2000).
WNT signalling molecules act in axis formation in the diploblastic metazoan Hydra. Nature 407, 186 –189.
Hobmayer, E., Holstein, T. W., and David, C. N. (1990). Tentacle
morphogenesis in hydra. I. The role of head activator. Development 109, 887– 895.
Hoffmeister, S. A. (1989). Action of foot activator on growth and
differentiation of cells in hydra. Dev. Biol. 133, 254 –261.
Hoffmeister, S. A. H. (1996). Isolation and characterization of two
new morphogenetically active peptides from Hydra vulgaris.
Development 122, 1941–1948.
Hoffmeister, S. A. H., and Schaller, H. C. (1987). Head activator and
head inhibitor are signals for nerve cell differentiation in hydra.
Dev. Biol. 122, 72–77.
Hoffmeister-Ullerich, S. A. (2001). The foot formation stimulating
peptide pedibin is also involved in patterning of the head in
hydra. Mech. Dev. 106, 37– 45.
Holstein, T., and David, C. N. (1986). The properties of nerve cell
precursors in hydra. Dev. Biol. 115, 18 –26.
Holstein, T., Schaller, C. H., and David, C. N. (1986). Nerve cell
differentiation in hydra requires two signals. Dev. Biol. 115,
9 –17.
Holstein, T. W., Hobmayer, E., and David, C. N. (1991). Pattern of
epithelial cell cycling in hydra. Dev. Biol. 148, 602– 611.
Howell, B. W., and Herz, J. (2001). The LDL receptor gene family:
Signaling functions during development. Curr. Opin. Neurobiol.
11, 74 – 81.
Jacobsen, L., Madsen, P., Jacobsen, C., Nielsen, M. S., Gliemann, J.,
and Petersen, C. M. (2001). Activation and functional characterization of the mosaic receptor SorLA/LR11. J. Biol. Chem. 276,
22788 –22796.
Jacobsen, L., Madsen, P., Moestrup, S. K., Lund, A. H., Tommerup,
N., Nykjaer, A., Sottrup-Jensen, L., Gliemann, J., and Petersen,
C. M. (1996). Molecular characterization of a novel human
hybrid-type receptor that binds the alpha2-macroglobulin
receptor-associated protein. J. Biol. Chem. 271, 31379 –31383.
Javois, L. C., and Tombe, V. K. (1991). Head activator does not
qualitatively alter head morphology In regenerates of Hydra
oligactis. Roux’s Arch. Dev. Biol. 199, 402– 408.
Johnson, E. M., and Chun, Y. H. (1989). In vitro differential
developmental toxicity of vitamin A congeners. Teratology 39,
349 –361.
© 2002 Elsevier Science (USA). All rights reserved.
217
Developmental Signaling in Hydra
Kaloulis, K. (2000). Molecular basis of morphogenetic events in
hydra: Study of the CREB and Hedgehog pathways during budding and regeneration. Ph.D. Thesis, University of Geneva.
Kim, J., Kim, W., and Cunningham, C. W. (1999). A new perspective on lower metazoan relationships from 18S rDNA sequences.
Mol. Biol. Evol. 16, 423– 427.
Knight, R. D., and Shimeld, S. M. (2001). Identification of conserved
C2H2 zinc-finger gene families in the Bilateria. Genome Biol. 2,
RESEARCH0016.
Koizumi, O., and Bode, H. R. (1986). Plasticity in the nervous
system of adult hydra. I. The position-dependent expression of
FMRFamide-like immunoreactivity. Dev. Biol. 116, 407– 421.
Kroiher, M., Miller, M. A., and Steele, R. E. (2001). Deceiving
appearances: Signaling by “dead” and “fractured” receptor
protein-tyrosine kinases. Bioessays 23, 69 –76.
Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K.,
Nagai, R., Oda, H., Kuwaki, T., Cao, W. H., Kamada, N., et al.
(1994). Elevated blood pressure and craniofacial abnormalities in
mice deficient in endothelin-1. Nature 368, 703–710.
Kuwabara, P. E., Lee, M. H., Schedl, T., and Jefferis, G. S. (2000). A
C. elegans patched gene, ptc-1, functions in germ-line cytokinesis. Genes Dev. 14, 1933–1944.
Lelong, C., Mathieu, M., and Favrel, P. (2001). Identification of new
bone morphogenetic protein-related members in invertebrates.
Biochimie 83, 423– 426.
Lenhoff, H. M. (1983). “Hydra: Research Methods.” Plenum Press,
New York.
Leontovich, A. A., Zhang, J., Shimokawa, K., Nagase, H., and
Sarras, M. P., Jr. (2000). A novel hydra matrix metalloproteinase
(HMMP) functions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot
process. Development 127, 907–920.
Lillien, L. (1995). Changes in retinal cell fate induced by overexpression of EGF receptor. Nature 377, 158 –162.
Littlefield, C. L. (1985). Germ cells in Hydra oligactis males. I.
Isolation of a subpopulation of interstitial cells that is developmentally restricted to sperm production. Dev. Biol. 112, 185–
193.
Littlefield, C. L. (1991). Cell lineages in Hydra: Isolation and
characterization of an interstitial stem cell restricted to egg
production in Hydra oligactis. Dev. Biol. 143, 378 –388.
Littlefield, C. L. (1994). Cell– cell interactions and the control of
sex determination in hydra. Semin. Dev. Biol. 5, 13–20.
Lohmann, J. U., and Bosch, T. C. (2000). The novel peptide HEADY
specifies apical fate in a simple radially symmetric metazoan.
Genes Dev. 14, 2771–2777.
Lohmann, J. U., Endl, I., and Bosch, T. C. (1999). Silencing of
developmental genes in Hydra. Dev. Biol. 214, 211–214.
Maden, M. (2000). The role of retinoic acid in embryonic and
post-embryonic development. Proc. Nutr. Soc. 59, 65–73.
Mao, B., Wu, W., Li, Y., Hoppe, D., Stannek, P., Glinka, A., and
Niehrs, C. (2001). LDL-receptor-related protein 6 is a receptor for
Dickkopf proteins. Nature 411, 321–325.
Marcum, B. A. (1983). Culturing epithelial Hydra. In “Hydra:
Research Methods” (H. M. Lenhoff, Ed.), pp. 287–290. Plenum
Press, New York.
Marcum, B. A., and Campbell, R. D. (1978). Development of Hydra
lacking nerve and interstitial cells. J. Cell Sci. 29, 17–33.
Marshall, C. J. (1995). Specificity of receptor tyrosine kinase
signaling: Transient versus sustained extracellular signalregulated kinase activation. Cell 80, 179 –185.
Martin, V. J., Littlefield, C. L., Archer, W. E., and Bode, H. R. (1997).
Embryogenesis in hydra. Biol. Bull. 192, 345–363.
Massague, J. (1998). TGF-beta signal transduction. Annu. Rev.
Biochem. 67, 753–791.
Medina, M., Collins, A. G., Silberman, J. D., and Sogin, M. L.
(2001). Evaluating hypotheses of basal animal phylogeny using
complete sequences of large and small subunit rRNA. Proc. Natl.
Acad. Sci. USA 98, 9707–9712.
Miller, M. A., Malik, I. A., Shenk, M. A., and Steele, R. E. (2000).
The Src/Csk regulatory circuit arose early in metazoan evolution. Oncogene 19, 3925–3930.
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.
Minobe, S., Fei, K., Yan, L., Sarras, M., Jr., and Werle, M. (2000).
Identification and characterization of the epithelial polarity
receptor “Frizzled” in Hydra vulgaris. Dev. Genes Evol. 210,
258 –262.
Mokady, O., and Buss, L. W. (1996). Transmission genetics of
allorecognition in Hydractinia symbiolongicarpus (Cnidaria:
Hydrozoa). Genetics 143, 823– 827.
Müller, W. A. (1984). Retinoids and pattern formation in a hydroid.
J. Embryol. Exp. Morphol. 81, 253–271.
Müller, W. A. (1989). Diacylglycerol-induced multihead formation
in Hydra. Development 105, 306 –316.
Müller, W. A. (1990). Ectopic head and foot formation in Hydra:
Diacylglycerol-induced increase in positional value and assistance of the head in foot formation. Differentiation 42, 131–143.
Mumm, J. S., and Kopan, R. (2000). Notch signaling: from the
outside in. Dev. Biol. 228, 151–165.
Nasevicius, A., and Ekker, S. C. (2000). Effective targeted gene
“knockdown” in zebrafish. Nat. Genet. 26, 216 –220.
Niehrs, C. (1999). Head in the WNT: The molecular nature of
Spemann’s head organizer. Trends Genet. 15, 314 –319.
Nothacker, H. P., and Grimmelikhuijzen, C. J. (1993). Molecular
cloning of a novel, putative G protein-coupled receptor from sea
anemones structurally related to members of the FSH, TSH,
LH/CG receptor family from mammals. Biochem. Biophys. Res.
Commun. 197, 1062–1069.
Owen, G. I., and Zelent, A. (2000). Origins and evolutionary
diversification of the nuclear receptor superfamily. Cell. Mol.
Life Sci. 57, 809 – 827.
Pawson, T., and Bernstein, A. (1990). Receptor tyrosine kinases:
Genetic evidence for their role in Drosophila and mouse development. Trends Genet. 6, 350 –356.
Peterson, R. T., Link, B. A., Dowling, J. E., and Schreiber, S. L.
(2000). Small molecule developmental screens reveal the logic
and timing of vertebrate development. Proc. Natl. Acad. Sci.
USA 97, 12965–12969.
Phiel, C. J., and Klein, P. S. (2001). Molecular targets of lithium
action. Annu. Rev. Pharmacol. Toxicol. 41, 789 – 813.
Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J., and Skarnes,
W. C. (2000). An LDL-receptor-related protein mediates Wnt
signalling in mice. Nature 407, 535–538.
Reber-Muller, S., Studer, R., Muller, P., Yanze, N., and Schmid, V.
(2001). Integrin and talin in the jellyfish podocoryne carnea. Cell
Biol. Int. 25, 753–769.
Reidling, J. C., Miller, M. A., and Steele, R. E. (2000). Sweet Tooth,
a novel receptor protein-tyrosine kinase with C-type lectin-like
extracellular domains. J. Biol. Chem. 275, 10323–10330.
© 2002 Elsevier Science (USA). All rights reserved.
218
Robert Steele
Robertson, H. M. (1997). Multiple Mariner transposons in flatworms and hydras are related to those of insects. J. Hered. 88,
195–201.
Rubanyi, G. M., and Polokoff, M. A. (1994). Endothelins: Molecular
biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol. Rev. 46, 325– 415.
Ruvkun, G., and Hobert, O. (1998). The taxonomy of developmental control in Caenorhabditis elegans. Science 282, 2033–2041.
Samuel, G., Miller, D., and Saint, R. (2001). Conservation of a
DPP/BMP signaling pathway in the nonbilateral cnidarian Acropora millepora. Evol. Dev. 3, 241–250.
Sarras, M. P., Jr., 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.
Sarras, M. P., Jr., Meador, D., and Zhang, X. M. (1991). Extracellular
matrix (mesoglea) of Hydra vulgaris. II. Influence of collagen and
proteoglycan components on head regeneration. Dev. Biol. 148,
495–500.
Sarras, M. P., Jr., 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.
Sarras, M. P., Jr., 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.
Schaller, H., and Gierer, A. (1973). Distribution of the headactivating substance in hydra and its localization in membranous
particles in nerve cells. J. Embryol. Exp. Morphol. 29, 39 –52.
Schaller, H. C. (1973). Isolation and characterization of a lowmolecular-weight substance activating head and bud formation
in hydra. J. Embryol. Exp. Morphol. 29, 27–38.
Schaller, H. C. (1976a). Action of the head activator as a growth
hormone in hydra. Cell Differ. 5, 1–11.
Schaller, H. C. (1976b). Action of the head activator on the
determination of interstitial cells in hydra. Cell Differ. 5, 13–25.
Schaller, H. C., and Bodenmüller, H. (1981). Isolation and amino
acid sequence of a morphogenetic peptide from hydra. Proc. Natl.
Acad. Sci. USA 78, 7000 –7004.
Schaller, H. C., Grimmelikhuijzen, C. J. P., and Schmidt, T. (1983).
Assay and isolation of substances controlling morphogenesis in
Hydra. In “Hydra: Research Methods” (H. M. Lenhoff, Ed.), pp.
311–324. Plenum Press, New York.
Schaller, H. C., Hoffmeister, S. A., and Dubel, S. (1989). Role of the
neuropeptide head activator for growth and development in
hydra and mammals. Development 107 (Suppl.), 99 –107.
Schaller, H. C., Rau, T., and Bode, H. (1980). Epithelial cells in
nerve-free hydra produce morphogenetic substances. Nature 283,
589 –591.
Schawaller, M., Schenck, K., Hoffmeister, S. A., Schaller, H., and
Schaller, H. C. (1988). Production and characterization of monoclonal antibodies recognizing head activator in precursor form
and immunocytochemical localization of head activator precursor and head activator peptide in the neural cell line NH15-CA2
and in hydra. Differentiation 38, 149 –160.
Semenov, M. V., Tamai, K., Brott, B. K., Kuhl, M., Sokol, S., and He,
X. (2001). Head inducer Dickkopf-1 is a ligand for Wnt coreceptor
LRP6. Curr. Biol. 11, 951–961.
Shilo, B. Z. (1992). Roles of receptor tyrosine kinases in Drosophila
development. FASEB J. 6, 2915–2922.
Smith, K. M., Gee, L., and Bode, H. R. (2000). HyAlx, an aristalessrelated gene, is involved in tentacle formation in hydra. Development 127, 4743– 4752.
Steele, R. E., Lieu, P., Mai, N. H., Shenk, M. A., and Sarras, M. P.,
Jr. (1996). Response to insulin and the expression pattern of a
gene encoding an insulin receptor homologue suggest a role for
an insulin-like molecule in regulating growth and development
in hydra. Dev. Genes Evol. 206, 247–259.
Sternberg, P. W., and Han, M. (1998). Genetics of RAS signaling in
C. elegans. Trends Genet. 14, 466 – 472.
Sternberg, P. W., Lesa, G., Lee, J., Katz, W. S., Yoon, C., Clandinin,
T. R., Huang, L. S., Chamberlin, H. M., and Jongeward, G. (1995).
LET-23-mediated signal transduction during Caenorhabditis elegans development. Mol. Reprod. Dev. 42, 523–528.
Sugiyama, T. (1983). Isolating Hydra mutants by sexual inbreeding.
In “Hydra: Research Methods” (H. M. Lenhoff, Ed.), pp. 211–221.
Plenum Press, New York.
Sugiyama, T., and Fujisawa, T. (1977). Genetic analysis of developmental mechanisms in hydra. III. Characterization of a
regeneration-deficient strain. J. Embryol. Exp. Morphol. 42,
65–77.
Takahashi, T., Koizumi, O., Ariura, Y., Romanovitch, A., Bosch,
T. C., Kobayakawa, Y., Mohri, S., Bode, H. R., Yum, S., Hatta, M.,
and Fujisawa, T. (2000). A novel neuropeptide, Hym-355, positively regulates neuron differentiation in Hydra. Development
127, 997–1005.
Takahashi, T., Muneoka, Y., Lohmann, J., Lopez de Haro, M. S.,
Solleder, G., Bosch, T. C., David, C. N., Bode, H. R., Koizumi, O.,
Shimizu, H., Hatta, M., Fujisawa, T., and Sugiyama, T. (1997).
Systematic isolation of peptide signal molecules regulating development in hydra: LWamide and PW families. Proc. Natl.
Acad. Sci. USA 94, 1241–1246.
Takano, J., and Sugiyama, T. (1985). Genetic analysis of developmental mechanisms in hydra. XVI. Effect of food on budding and
developmental gradients in a mutant strain L4. J. Embryol. Exp.
Morphol. 90, 123–138.
Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama,
Y., Hess, F., Saint-Jeannet, J. P., and He, X. (2000). LDL-receptorrelated proteins in Wnt signal transduction. Nature 407, 530 –
535.
Tan, P. B., and Kim, S. K. (1999). Signaling specificity: The
RTK/RAS/MAP kinase pathway in metazoans. Trends Genet.
15, 145–149.
Technau, U., and Bode, H. R. (1999). HyBra1, a Brachyury homologue, acts during head formation in Hydra. Development 126,
999 –1010.
Trembley, A. (1744). “Mémoires, pour servir à l’historie d’un genre
de polypes d’eau douce, à bras en forme de cornes.” Jean and
Herman Verbeek, Leiden.
Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P., and Sharp,
P. A. (1999). Targeted mRNA degradation by double-stranded
RNA in vitro. Genes Dev. 13, 3191–3197.
Wainright, P. O., Hinkle, G., Sogin, M. L., and Stickel, S. K. (1993).
Monophyletic origins of the metazoa: An evolutionary link with
fungi. Science 260, 340 –342.
Wehrli, M., Dougan, S. T., Caldwell, K., O’Keefe, L., Schwartz, S.,
Vaizel-Ohayon, D., Schejter, E., Tomlinson, A., and DiNardo, S.
(2000). arrow encodes an LDL-receptor-related protein essential
for Wingless signalling. Nature 407, 527–530.
Wodarz, A., and Nusse, R. (1998). Mechanisms of Wnt signaling in
development. Annu. Rev. Cell Dev. Biol. 14, 59 – 88.
© 2002 Elsevier Science (USA). All rights reserved.
219
Developmental Signaling in Hydra
Yanagisawa, H., Yanagisawa, M., Kapur, R. P., Richardson, J. A.,
Williams, S. C., Clouthier, D. E., de Wit, D., Emoto, N., and
Hammer, R. E. (1998). Dual genetic pathways of endothelinmediated intercellular signaling revealed by targeted disruption of
endothelin converting enzyme-1 gene. Development 125, 825– 836.
Yao, T. (1945). Studies on the organizer problem in Pelmatohydra
oligactis. I. The induction potency of the implants and the nature
of the induced hydranth. J. Exp. Biol. 21, 147–150.
Yee, J.-K., Friedmann, T., and Burns, J. C. (1994). Generation of
high-titer pseudotyped retroviral vectors with very broad host
range. Methods Cell Biol. 43, 99 –112.
Zhang, J., Leontovich, A., and Sarras, M. P., Jr. (2001). Molecular
and functional evidence for early divergence of an endothelin-
like system during metazoan evolution: Analysis of the Cnidarian, hydra. Development 128, 1607–1615.
Zhang, X., Hudson, B. G., and Sarras, M. P., Jr. (1994). Hydra cell
aggregate development is blocked by selective fragments of
fibronectin and type IV collagen. Dev. Biol. 164, 10 –23.
Zhang, X., and Sarras, M. P., Jr. (1994). Cell-extracellular matrix
interactions under in vivo conditions during interstitial cell
migration in Hydra vulgaris. Development 120, 425– 432.
© 2002 Elsevier Science (USA). All rights reserved.
Received for publication September
Revised May
Accepted May
Published online July
10,
10,
28,
22,
2001
2002
2002
2002