DEVELOPMENTAL DYNAMICS 226:225–236, 2003
REVIEWS
A PEER REVIEWED FORUM
Head Regeneration in Hydra
Hans R. Bode*
Hydra, a primitive metazoan, has a simple structure consisting of a head, body column, and foot aligned along a single
oral–aboral axis. The body column has a high capacity for regeneration of both the head and foot. Because of the
tissue dynamics that take place in adult Hydra, the processes governing axial patterning are continuously active to
maintain the form of the animal. Regeneration in hydra is morphallactic and closely related to these axial patterning
processes. As might be expected, analysis at the molecular level indicates that the same set of genes are involved in
head regeneration and the maintenance of the head in the context of the tissue dynamics of the adult. The genes
analyzed so far play roles in axial patterning processes in bilaterians. Developmental Dynamics 226:225–236, 2003.
© 2003 Wiley-Liss, Inc.
Key words: Hydra; head regeneration; axial patterning; organizer
Received 20 August 2002; Accepted 14 October 2002
INTRODUCTION
Hydra, a member of the Cnidaria, is
a primitive metazoan with a simple
body plan. It consists of a single oral–
aboral axis with radial symmetry. The
structures along the axis are a head,
a body column, and a foot. As a
consequence of its tissue dynamics,
the animal has an extensive capacity for the morphallactic regeneration of head and foot. In the following, the structure, tissue dynamics,
regeneration phenomena, processes governing axial patterning as
well as what is known about the molecular basis of regeneration will be
described. The emphasis will be on
head regeneration, as that has
been studied in more detail.
STRUCTURE AND TISSUE
DYNAMICS OF Hydra
The simple body plan of a Hydra is
shown in Figure 1. The single axis
consists of a cylindrical shell sur-
rounding a gastric cavity that extends throughout the body column.
The head consists of two parts. The
apical part is the hypostome, or
mouth region, whereas the basal part
is the tentacle zone from which a ring
of tentacles emerge. At the opposite
end, the body column ends in the
foot, or basal disk, with which the animal attaches itself to surfaces in its
freshwater environment.
The overall structure of a hydra is
based on two epithelial layers, the
ectoderm and endoderm (Fig. 1).
Each layer is a single cell thick, which
extends throughout the animal. Between these two layers is a basement membrane called the mesoglea. The epithelial cells of each of
the two layers is a cell lineage consisting of stem cells in the body column and differentiated cells in the
extremities. All of the remaining cells
belong to a third cell lineage, the
interstitial cell lineage. These cells
are located in the spaces, or inter-
stices, among the epithelial cells of
both layers and are scattered
throughout the animal (not shown in
Fig. 1). These interstitial cells are multipotent stem cells that give rise to
four sets of differentiation products:
neurons, secretory cells, gametes,
and nematocytes, the stinging cells
that are typical of cnidarians.
When hydra are fed on a regular
basis, all of the epithelial cells in the
body column of both layers are continuously in the mitotic cycle (David
and Campbell, 1972; Campbell and
David, 1974). However, the animal
does not increase in size. Instead, tissue, as shown in Figure 1, is continuously displaced into the extremities
and sloughed (Campbell, 1967; Otto
and Campbell, 1977). It is also displaced onto developing buds,
which eventually detach from the
adult. Bud formation is hydra’s form
of asexual reproduction. Hence, the
tissues of an adult hydra are in a
steady state of production and loss.
Developmental Biology Center and Department of Developmental and Cell Biology, University of California, Irvine, California
*Correspondence to: Hans R. Bode, Department of Developmental and Cell Biology, University of California, Irvine, CA 92697.
E-mail: [email protected]
DOI 10.1002/dvdy.10225
© 2003 Wiley-Liss, Inc.
226 BODE
Fig. 1. Longitudinal cross-section of an
adult Hydra. The regions and the two tissue
layers are indicated. The protrusions from
the body column are early and later stages
of bud development: Hydra's asexual form
of reproduction. The arrows indicate the directions of tissue displacement. (Reprinted
with permission of the Editor of Integrative
and Comparative Biology.)
In a little more detail, tissue of both
layers in the upper part of the body
column is displaced into the tentacle zone, and then out along the
tentacles, and eventually sloughed
at the tentacle tips. Some of the
endodermal tissue is displaced into
the hypostome, moving progressively toward the tip where it is
sloughed (Dubel et al., 1987). In contrast, ectodermal tissue of the hypostome is generated by dividing epithelial cells at the base of the
hypostome, which are displaced
apically and shed (Dubel et al.,
1987). Similarly, tissue of both layers
displaced down the body column
ends up on the foot and is sloughed.
Loss at these extremities accounts
for ⬃15% of the tissue lost from the
animal (Otto and Campbell, 1977);
most of the tissue (⬃85%) is displaced onto developing buds.
The tissue dynamics reflect the nature of the three stem cell lineages.
For the ectoderm, the epithelial cells
of the body column are the stem
cells that are constantly in the mitotic cycle. As these cells are displaced across the tentacle zone/
tentacle border (see Fig. 4), they
cease dividing and differentiate into
the battery cells of the tentacle (Holstein et al., 1991). Similarly, when the
ectoderm is displaced basally crossing the peduncle/foot border (Fig.
1), onto the foot, the ectodermal
epithelial cells cease dividing and
differentiate into cells of the foot
(Holstein et al., 1991). The epithelial
cells of the endoderm undergo a
similar behavior. All cells in the body
column are in the mitotic cycle.
When displaced apically across the
tentacle zone/tentacle border, or
basally across the peduncle/basal
disk border, they cease dividing and
differentiate.
Thus, both epithelial cell lineages
have the characteristics of a stem
cell lineage. In one aspect, they differ from the typical stem cell lineages found in bilaterians. Usually
the activity of stem cells is to divide
to maintain their population as well
as producing cells that differentiate
and carry out a physiological function. However, the stem cells do not
have a physiological function. In
contrast, the epithelial cells of both
layers in the Hydra body column that
behave like epithelial stem cells as
they divide continuously also have a
physiological function. The endodermal epithelial cells act as digestive
cells, whereas the ectodermal epithelial cells function in the protective
manner of skin cells. This finding
could reflect the evolutionarily primitive state of Hydra. Plausibly during
early stages of metazoan evolution,
stem cells had both a stem cell function and a physiological function.
Later, in more advanced metazoans, these functions were separated.
The third lineage, the interstitial
cell lineage, is more similar to bilaterian stem cell lineages. The interstitial cells, the multipotent stem cells,
are scattered throughout the ectoderm of the body column (Bode,
1996). They act as true stem cells,
giving rise to cells of the four different classes of differentiation products (David and Murphy, 1977). The
population of each differentiation
product is also in a steady state of
production and loss. They arise from
the multipotent stem cells and are
displaced with the epithelial cells,
and eventually lost by sloughing at
an extremity or by displacement
onto a bud. Hence, the multipotent
interstitial cells are continuously in
the mitotic cycle to maintain their
own population size in the context of
the continuous expansion of the two
epithelia as well as the continuous
production of differentiation products to compensate for their loss at
the extremities.
REGENERATION IN Hydra
Regeneration Phenomena
The body column has quite an extraordinary capacity for the regeneration of a missing head or foot.
When a hydra is bisected in the
body column, the upper half will regenerate a foot at its basal end,
whereas the lower half will regenerate a head at its apical end (Fig.
2A). This finding occurs when the animal is bisected anywhere along the
upper 7/8ths of the body column.
Similarly, when a piece of the body
column is isolated, it will invariably
regenerate a head at the apical
end, and a basal disk, or foot, at the
basal end (Fig. 2B). That the head
and foot form at the apical and
basal ends of the isolated piece indicates that the tissue has a apical–
basal, or oral–aboral, polarity, which
is maintained after excision. One
other manipulation indicates a further aspect of this regeneration capacity. When an isolated body column is cut in half longitudinally (Fig.
2C), it rapidly heals into a narrow
cylinder, and regenerates a head
and foot both with smaller diameters. With time, each region regains,
or regenerates, its normal diameter.
This finding indicates there are
mechanisms for establishing a particular diameter-to-length ratio in
the body column.
Perhaps the most dramatic form
of regeneration in hydra is the ability
to form complete animals from aggregates of cells (Gierer et al., 1972).
When hydra are dissociated into a
suspension of viable cells and subsequently aliquots of these cells are
centrifuged into pellets, each pellet,
or aggregate, will develop into one
or more complete hydra. In a little
more detail, the cells of the aggregate sort out so that, within 24 hr, it
consists of a two-layered spherical
shell in which the two layers are the
ectoderm and endoderm of the
adult animal. Thereafter, heads
form, which subsequently organize
surrounding tissue into the body column and foot of a hydra, and detach from the remaining tissue. In
CHARACTERIZATION OF THE Hydra ORGANIZER 227
Fig. 2. Regeneration capacities of a Hydra. A: The regeneration of the missing extremity
on each half after bisection. B: The regeneration of both head and foot after isolation of a
piece of the body column. C: Regeneration of the extremities as well as the body column
after isolation of a longitudinal half of the body column. D: Regeneration of complete
animal from a small fraction of the body column. Dark head and foot regions arose by
regeneration.
Fig. 3. Morphologic changes during head regeneration after decapitation.
aggregates, the patterning processes for head formation, axial patterning, and foot formation start de
novo, as there is no polarity in the
tissue of the spherical shell.
Another aspect is the size of the
piece capable of regeneration. Excision of a piece representing approximately 5% of the body column
results in the regeneration of complete Hydra with normal proportions
(Fig. 2D; Bode and Bode, 1980). Still
smaller pieces corresponding to approximately 1% of the body column
will regenerate both head and foot,
although the relative sizes of head
and foot compared with the body
column are not normal (Bode and
Bode, 1980, Shimizu et al., 1993). This
finding again reflects an intrinsic po-
larity in the tissue, and a highly developed capacity for proportion
regulation.
This extensive regeneration capacity is limited to the body column.
An isolated head will not regenerate
a foot, and an isolated foot or the
isolated foot ⫹ lower eighth of the
body column will not regenerate a
head. Similarly, an isolated tentacle
simply disintegrates with time. Thus,
the regeneration capacity is limited
to the body column where the stem
cells of all three lineages are undergoing cell division.
Characteristics of Regeneration
The head regeneration process is
quite rapid. After bisection of the
body column, head regeneration in
the lower part consists of the following (Fig. 3). The epithelia at the
wounded upper end of the lower
piece stretch to cover and close the
wound, which takes place in 3 to 6
hr. Within 30 to 36 hr, tentacles begin
to emerge, and by 48 to 72 hr, a fully
regenerated head has formed. Foot
regeneration is a similar process in
that the epithelia stretch to close the
wound, and then a foot begins to
form. As measured by the reappearance of a foot-specific peroxidase
(Hoffmeister and Schaller, 1985) as
well as the ability of the piece of
tissue to stick to a surface, a foot
regenerates in approximately 30 hr.
Regeneration processes are of
two kinds. In epimorphic regeneration, cell division takes place at and
near the cut surface to provide tissue from which the removed structure regenerates. The regenerated
structure, such as a limb, is the same
size as the normal limb. In contrast,
during morphallactic regeneration,
cell division is unnecessary as the remaining tissue is remodeled to build
the missing structures. In Hydra, regeneration is morphallactic, as illustrated by the regeneration of a
complete, albeit much smaller, animal from a piece of the body column. That cell division is unnecessary
for regeneration in Hydra was demonstrated by blocking cell division
with ␥-irradiation (Hicklin and Wolpert, 1973) or hydroxyurea (Cummings and Bode, 1984), bisecting
the body column, and allowing regeneration to occur. The rates and
extent of regeneration in pieces in
which cell division was blocked were
very similar in treated and untreated
controls (Cummings and Bode,
1984).
Another issue concerns which of
the three cell lineages are responsible for head and foot regeneration,
and what roles do they play. Obviously, the epithelial cells of both layers are involved. However, the interstitial cell lineage is not required. The
interstitial cell lineage can be removed from hydra in several ways.
Because of the shorter cell cycle
time [1 day vs. 3 days], treatment
with hydroxyurea (Cummings and
Bode, 1984) or colchicine (Campbell, 1976) selectively reduces and
228 BODE
eventually removes the interstitial
cell population. Thereafter, the tissue
displacement patterns coupled with
sloughing at the extremities leads to
the loss of all the interstitial cell differentiation products. Such animals,
termed “epithelial animals” can be
maintained indefinitely by handfeeding, and will reproduce by budding as do normal animals. Epithelial
animals maintained for over a year,
when bisected, will regenerate a
head and foot on the lower and upper parts, respectively, as do normal
animals (Marcum and Campbell,
1978). Similar results were obtained
with nf-1, a mutant of Hydra magnipapillata devoid of cells of the interstitial cell lineage (Sugiyama and
Fujisawa, 1978). When bisected, animals of this strain regenerated a
head at the same rate as a strain
containing interstitial cells.
Although both epithelial lineages
are required for head and foot regeneration simply for structural purposes, the question arises as to
whether the control, or patterning,
of the head during head regeneration is controlled by one or both layers. A similar question exists for foot
regeneration. Smid and Tardent
(1982) examined this issue by removing head and foot, separating the
two epithelial layers, and generating
chimeras by recombining the two
layers either with the same headfoot polarity, or with an opposite polarity. Those chimeras with the same
head-foot polarity all regenerated
normally. However, the majority of
those chimeras in which the ectoderm and endoderm had opposite
polarities, formed a new axis that
was perpendicular to the original
axis, indicating that neither layer
alone controlled where head and
foot formed during regeneration. Instead, both layers play a role in determining the head/foot polarity.
Do the two layers provide the same
or different information during head
or foot regeneration? This question
was examined by Zeretzke and Berking (2002) who made use of mh-1, a
mutant of Hydra magnipapillata that
forms ectopic heads along the body
column (Sugiyama and Fujisawa,
1977). In addition, upon bisection,
mh-1 animals regenerate a foot at a
slower rate than does the 105 strain,
which is the wild-type. By making ectoderm/endoderm chimeras [same
head–foot polarity for both layers),
they generated mh-1 ecto/105
endo and 105 ecto/mh-1 endo chimeras. They found that the mh-1
ecto/105 endo chimeras formed ectopic heads along the body column,
whereas the 105 ecto/mh-1 endo
chimeras regenerated feet at a reduced rate. These results suggest
that, in this situation, the ectoderm
influences head formation, whereas
the endoderm affects foot formation. Whether this is generally true,
will require a similar analysis using
mutants with alterations in different
characteristics of head and foot.
In summary, the body column has
a formidable capacity for head and
foot regeneration, which occurs in a
morphallactic manner. Both epithelial cell lineages are required for regeneration, and the two lineages
may play different roles. The interstitial cell lineage is not required for
either head or foot regeneration.
PATTERNING PROCESSES
GOVERNING AXIAL PATTERNING
IN Hydra
Because an adult Hydra is in a
steady state of production and loss
of tissue coupled with continual displacement of tissue along the body
axis, the processes governing axial
patterning need to be constantly
active to maintain the morphology
of the animal. These same processes
are also involved in head regeneration.
A large body of transplantation
experiments have demonstrated
that the central element of the axial
patterning process is a morphogenetic gradient that has a maximum
value in the head. This gradient has
been referred to as a gradient of
positional value (Wolpert et al.,
1971), the head activation gradient
(MacWilliams, 1983b], or the source
density gradient (Gierer and Meinhardt, 1972). The term head activation (HA) gradient will be used here.
More recently, it has become
clear that the gradient consists of
two components. One is an organizer located in the hypostome,
whereas the other is the HA gradient, which is confined to the body
column. The hypostome, or more
specifically the head organizer in the
hypostome, has the typical characteristic of an organizer in that it can
induce a second axis when transplanted to the body column of a
host hydra (e.g., Browne, 1909; MacWilliams, 1983b; Broun and Bode,
2002). This second axis consists of a
head and body column but no foot.
When a piece of the upper part of
the body column is transplanted to
a lower axial location in a second
animal, it can also form a second
axis consisting of head and body
column. However, this axis is formed
by self-organization of the transplanted piece and not by induction
of host tissue (Broun and Bode,
2002). This distinction is supported by
the finding that a piece of body column tissue similar in size to the hypostome will not induce or form a second axis upon transplantation to a
host body column (Yao, 1945; Broun
and Bode, 2002). Hence, the organizing capacity is restricted to the
hypostome.
The organizer capacity of the hypostome and the head formation
capacity, or HA, of the body column
differ in two other ways. (1) By using
a transplantation experiment designed to examine the stability of HA
in the body column, MacWilliams
(1983b) found that the HA associated with a regenerating head had
a half-life of 12 hr compared with the
36-hr half-life of the HA of the body
column. This “unstable head activation” (MacWilliams, 1983b) in the regenerating head corresponds to the
organizer region, indicating that the
organizer is intrinsically less stable
than is the HA gradient. (2) Treatment with 0.5–1 mM LiCl lowers the
level of the HA gradient in the body
column (Hassel and Berking, 1990)
but has no effect on the organizer
(Broun and Bode, 2002).
The head organizer transmits two
signals to the body column. One,
which is presumably graded down
the body axis, sets up the HA gradient
in the body column (e.g., Wilby and
Webster, 1970a; Broun and Bode,
2002). The other is a graded distribution of a signal, termed head inhibition (HI), which prevents body column
tissue from undergoing head formation (e.g., Wilby and Webster, 1970b;
CHARACTERIZATION OF THE Hydra ORGANIZER 229
Fig. 4. Axial location of components of
the axial patterning process governing
head formation. HO, the head organizer is
located in the hypostome; HA, head activation gradient; HI, head inhibition gradient.
MacWilliams, 1983a). Most likely, this
signal is a small molecule, as blocking
gap junction communication between epithelial cells in the body column also blocked transmission of HI
(Fraser et al., 1987).
In the context of the tissue dynamics of an adult Hydra, these elements
probably behave in the following
manner to maintain the steady state
morphology of the adult. As the tissue of the hypostome is continuously
displaced from its base to its tip and
sloughed, the head organizer is continuously undergoing self-renewal.
At the same time, the organizer continuously produces and transmits the
signals for HA and HI to the body
column to maintain the two gradients in a steady state condition (Fig.
4). As tissue is displaced up the body
column, it is exposed to a higher
concentration of the head activation signal, which is translated into a
higher level of HA. When it reaches
the apical end of the body column,
the level of HA surpasses that of HI
and the tissue is committed to head
formation. Similarly, as tissue is displaced down the body columna, a
point is reached where [HA] ⬎ [HI]
and head formation is initiated to
begin the process of bud formation.
The reaction– diffusion model described by Gierer and Meinhardt
(1972) provides a mechanism that
can explain these results. Briefly, it
consists of two components: an activator (A) and an inhibitor (I). The
activator has a short diffusion range,
and is produced autocatalytically.
The I, which is produced cross-catalytically by the activator has a long
diffusion range. When [I] ⬎ [A], no
autocatalysis of the activator occurs, and the activator slowly decays. If [A] ⬎ [I], autocatalysis occurs, raising the activator beyond a
threshold level, resulting in the formation of the structure specified by
the activator, such as the head. The
autocatalytic character of the activator is useful in the dynamic context of the hypostomal tissue. In
addition, through an unspecified
mechanism, the activator sets up a
gradient, termed the source density
gradient, along the body column,
which is equivalent to the HA gradient. The source density gradient and
the head inhibition gradient differ in
shape because of the longer range
diffusion of the inhibitor as well as the
inhibitor having a shorter half-life
(see Fig. 4).
In a more refined version of the
model, which was in part stimulated
by the expression patterns of specific genes, Meinhardt (1993) separated the head into its two components, the hypostome and the
tentacle zone ⫹ tentacles. Instead
of a single activator, he introduced
a hypostome activator, a tentacle
activator, and an inhibitor for each
activator. As described later, this
model fits some of the molecular
data better.
Application of the Axial
Patterning Processes to Head
Regeneration
Head regeneration can be explained in terms of these axial patterning processes. HI is unstable with
a half-life of 2–3 hr (MacWilliams,
1983a). Although unstable, no
heads form in the upper two-thirds of
the body column because HI is continuously produced in the head organizer in the hypostome and trans-
mitted down the column. Upon
bisection, HI rapidly decays so that,
by 2 hr after bisection, the level of
HA as measured in transplantation
experiments, begins to rise at the
apical end of the lower half, indicating that [HA] ⬎ [HI]. By 8 hr, the HA
level has reached a maximum level
(MacWilliams, 1983b). As the rising
HA is the unstable HA described by
MacWilliams (1983b) and not the HA
of the stable HA gradient, this result
can be interpreted to mean that the
head organizer has been formed in
the future hypostome in the 8 hr after bisection. That a fall in HI is critical
was also demonstrated by Achermann and Sugiyama (1985). By using
reg-16, a mutant strain that regenerates a head in only 10 –20% of the
cases after bisection, they showed
that this mutant had a higher level of
HI, which did not fall rapidly. Furthermore, the HA gradient was lower
than in the wild-type, so that the
necessary condition for regeneration of [HA] ⬎ [HI] occurred infrequently.
Because the level of HI falls rapidly
throughout the lower half after bisection, one might expect unstable
HA to rise throughout the piece.
However, it does not. The level of HA
rises rapidly only at the apical tip
and only much more slowly throughout the rest of the lower half (MacWilliams, 1983a). The localized rise at
the apical end is probably due to
the additional factor of an injury
caused by bisection (MacWilliams,
1983b). This view is supported by the
finding that, if decapitated reg-16
animals that did not regenerate a
head were reinjured at the apical
end, almost all of them underwent
head regeneration (Kobatake and
Sugiyama, 1989). Thus, the higher
level of HA at the apical end coupled with the injury effect could lead
to the rapid formation of the head
organizer, which would produce
and transmit HI to prevent head formation from occurring elsewhere
along the body column. Of these
two factors, the more important one
is most likely the level of HA, because
simply injuring the body column
does not lead to head formation.
After formation of the head organizer, the regenerating head is divided into two regions, the hypos-
230 BODE
tome and the tentacle zone.
Subsequently, morphogenetic processes take place leading to the formation of a ring of tentacles in the
tentacle zone as well as the domelike form of the hypostome.
MOLECULAR COMPONENTS
AND MECHANISMS
UNDERLYING HEAD
REGENERATION
Because the processes governing
head regeneration are similar, or the
same as, those involved in maintaining the apical part of the axial pattern in the adult, one would expect
a similar set of genes to be involved.
Furthermore, most, if not all, of these
genes would also be involved in bud
formation. Thus, the overall strategy
has been to isolate genes from
whole animals, focusing on those
that are expressed in both deuterostomes and protostomes. Most likely,
such genes first appeared in the diploblasts, the evolutionary ancestors
of the bilaterians, which would include the cnidarians. This approach
has yielded homologues of bilaterian transcription factors, receptors,
signal transduction pathway components, as well as components of
basement membranes. As many
have been shown to participate in
head regeneration, a subset will be
discussed. A second approach,
which will be described below, has
focused on the isolation and characterization of peptides that act as
signalling molecules.
Wnt Pathway and the Head
Organizer
Because the head consists of two
parts, the hypostome and the tentacle zone, from which the tentacles
emerge, these two regions must be
patterned during head regeneration. The first issue is the development of the head organizer. As it is
set up within 8 hr at the apical end
after decapitation, genes expressed
during these early stages may involved in this process. A promising
set are the genes of the Wnt pathway. Homologues of several members, HyWnt, Hy␤-cat, and HyTcf,
have been isolated (Hobmayer et
al., 1996; Hobmayer et al., 2000),
and their expression patterns during
Fig. 5. Expression patterns of several head-specific genes in an adult hydra. The figure at
the upper left indicates the several parts of the head region. The different color in the
tentacles emphasizes the sharp border at the tentacle zone/tentacle boundary. Each
gene is indicated by a different color. The graded pattern for Tcf reflects the graded
distribution of expression, with a maximum at the tip.
head regeneration have been examined (Hobmayer et al., 2000). In
adult animals, all three are strongly
expressed in the hypostome with HyWnt confined to the very apex of the
hypostome, whereas the other two
are expressed throughout the hypostome, although more strongly at
the apex (Fig. 5). After bisection of
the body column, both HyTcf and
Hy␤-cat are strongly expressed all
over the regenerating cap within an
hour and HyWnt in the same pattern
within 3 hr (Hobmayer et al., 2000).
Other data support the view that
this pathway plays a role in the organizer. A critical element in the activity of the Wnt signalling pathway is
blocking the activity of GSK-3␤,
which leads to an elevated level of
␤-catenin. In turn, ␤-catenin coupled with Tcf activates the transcription of genes that play critical roles
in developmental processes (e.g.,
Peifer and Polakis, 2000). Treatment
of hydra with three different reagents that inhibit GSK-3␤ affect
head formation. (1) Treatment with
LiCl, which is known to block GSK-3␤
(e.g., Phiel and Klein, 2001) as well as
other enzymes, affects axial patterning in hydra. Treatment with 2 mM LiCl
leads to formation of ectopic tentacles and heads along the body column (Hassel et al., 1993). (2) Treatment with diacylglycerol leads to
ectopic head formation on the body
column (Muller, 1989). Diacylglycerol
stimulates the activity of PKC, which is
also known to inhibit GSK-3␤ activity
(Cook et al., 1996; Chen et al., 2000).
That HvPKC2, a hydra PKC gene, is
expressed in the same location as
the HyWnt gene in the apex of the
hypostomal endoderm (Fig. 5), provides additional support (Hassel et
al., 1998). (3) Using alsterpaullone,
an inhibitor known to specifically
block the activity of the GSK-3b enzyme (Leost et al., 2000), leads to the
formation of head organizer activity
in the body column (Broun et al.,
manuscript in preparation). All of
these results support the view that
the Wnt pathway plays a role in the
formation of the head organizer and
that the critical element is probably
␤-catenin.
Although these results are quite
clear, the processes are probably
more complex. If instead of using 2
mM LiCl one uses 0.5–1 mM LiCl, ectopic feet are formed (Hassel and
Berking, 1990), suggesting that Li ions
are interfering with a second pathway as well, plausibly the phosphatidylinasitol cycle.
Another aspect of the Wnt pathway supports the view that this pathway plays an important role in the
head organizer. As described in a
previous section, the continual turnover of the tissue of the hypostome
indicates the organizer is constantly
undergoing self-renewal, which could
be accomplished with a positive
CHARACTERIZATION OF THE Hydra ORGANIZER 231
feedback loop. This finding is also an
essential characteristic of the autocatalytic loop of the activator in the
reaction-diffusion model (Gierer and
Meinhardt, 1972). In Drosophila during wing disc development, the Wnt
pathway is known to act in just this
manner with a complex of Armadillo/␤-catenin and Tcf binding to the
Wnt promoter to activate Wnt production (Heslip et al., 1997). Thus, in
hydra, the cells expressing HyWnt,
which are on the verge of being lost
from the hypostome by sloughing,
would signal the neighboring basal
cells, thereby raising the level of the
␤-catenin protein. Subsequently,
these cells would begin the production of HyWnt. This positive feedback
loop could maintain the head organizer in a steady state in the context
of the tissue dynamics of the hypostome.
Other Genes Affecting Head
Patterning
Another gene that probably plays a
role in the patterning of the head
during head regeneration as well as
other forms of head formation is HyBra1, a hydra Brachyury homologue
(Technau and Bode, 1999). In the
adult animal, HyBra1 is expressed
throughout the hypostome, and
only there (Fig. 5). After bisection,
the gene appears as a small spot
within 3 hr in the apical tip and is
strongly expressed by 4 hr all over
the apical cap, which will eventually
form the hypostome. This pattern
suggests a role in hypostome formation and possibly in the organizer.
Four pieces of evidence support this
view (Technau and Bode, 1999). (1)
Upon decapitation of reg-16, the
head-regeneration–impaired
mutant HyBra1 is only expressed in the
small fraction of animals that explicitly regenerated a head. (2) By using
the transplantation experiment that
demonstrated that the half-life of
“unstable head activation,” or the
head organizer, is approximately 12
hr (MacWilliams, 1983b), it was
shown that the half-life of HyBra1 expression was also approximately 12
hr. (3) The time required for a regenerating tip to become committed to
forming a head increases the further
down the body column that the bi-
section takes place (Wilby and Webster, 1970a; MacWilliams, 1983b). The
same is true for the initial expression
of HyBra1. (4) Finally, it is possible that
expression of this gene may be correlated with a high level of HA but
not directly with the head organizer.
However, this is unlikely, because
raising the HA gradient in the body
column by treating animals with diacylglycerol or lowering it with a 1
mM LiCl treatment has no effect on
expression of this gene. In both
cases, the expression remains confined to the hypostome. Thus, HyBra1
probably plays a role in the patterning of the hypostome and, more
specifically, in the head organizer.
Another aspect of the patterning
of the head involves setting up the
two parts of the head: the hypostome and the tentacle zone. The behavior of five markers provides an
idea as to how this might take place
(see Fig. 5). Two of the markers are
genes expressed specifically in the
hypostome: HyBra1 and budhead, a
hydra HNF-3␤ homologue (Martinez
et al., 1997). Two other markers are
tentacle-specific: one is TS-19, a
monoclonal antibody that recognizes a tentacle-specific antigen
(Bode et al., 1988) and HyAlx, an
aristaless homologue [Smith et al.,
2000],which is expressed at the tentacle/tentacle zone border. After
decapitation, the two hypostome
markers are strongly expressed in the
regenerating apical cap by 4 and 8
hr, whereas the two tentacle markers are strongly expressed in the
same location somewhat later at
18 –20 hr. By 24 to 30 hr, the hypostomal markers remain strongly expressed in the apical cap, but the
two tentacle markers have begun to
shift toward a ring below the cap.
And, by 36 to 48 hr, when tentacle
buds begin to appear and develop
into tentacles, the tentacle markers
are mostly/completely expressed
only on the tentacles. This behavior
was also observed among the neurons in that tentacle-specific and hypostome-specific neurons are intermingled in the regenerating tip
during these early stages but separate at later stages (Mitgutsch et al.,
1999).
These changes in expression suggest that, during the early stages of
head regeneration, the tissue is becoming specified for head formation, but that the spatial distinctions
between hypostome and tentacle
zone have not been set up. With the
development of the head organizer
during the first 12 hr of head regeneration, the hypostome is becoming
defined. Subsequently, the lower
half of the regenerating head becomes committed to form the tentacle zone from which the tentacles
emerge.
The separation of the two layers is
also emphasized by the fifth marker,
Cngsc, the hydra homologue of
goosecoid (Broun et al., 1999). In the
adult head, Cngsc is expressed in a
narrow ring at the border between
the hypostome and tentacle zone
(Fig. 5). As the shifts in the tentacle
markers occur, expression of Cngsc
begins in the regenerating head. It
first appears at the apex by 24 hr,
and a day later its expression has
been displaced to the hypostome/
tentacle zone border where it remains. This behavior suggests the
gene is involved in setting up a border or boundary between the two
regions of the head. In Xenopus embryos, goosecoid is known to inhibit
brachyury expression (Artinger et al.,
1997). A similar interaction could be
occurring here with Cngsc, confining HyBra1 to the hypostome.
Meinhardt (1993) has provided a
formal explanation for these changing expression patterns with a modification of the reaction-diffusion
model for hydra (Gierer and Meinhardt, 1972). Instead of a single activator responsible for head formation, he has described a pair of
activators, one a hypostome activator, HypA, and the other a tentacle
activator, TA, each coupled with a
specific inhibitor, HypI and TI. The
two reaction– diffusion mechanisms
interact in that the HypA can block
the activity of the TA, which results in
the basal displacement of TA from
the regenerating tip to a position below the location of the HypA. This
displacement also results in the formation of a tentacle zone and a ring
of tentacles below the apex, which
will form the hypostome. Meinhardt
(1993) used the model to explain the
changes of expression of the TS-19
antigen during head regeneration
232 BODE
(Bode et al., 1988). Subsequently,
these changes were examined in
more detail and the model applied
(Technau and Holstein, 1995). It also
quite nicely explains the changes in
HyAlx expression during head regeneration by Smith et al. (2000).
A final question concerns the initiation of head regeneration. During
bud formation, a new axis is formed
with the subsequent development
of a complete new hydra. During regeneration, a missing component,
such as the head, is rebuilt as part of
an existing axis. All the genes described so far as well as several others have similar expression patterns
during both head regeneration and
bud formation. However, two genes,
CnOtx, a hydra otx homologue
(Smith et al, 1999), and Hymsx, a hydra msx homologue (McCord and
Bode, in preparation), have different
patterns. Hymsx is strongly expressed
during very early stages of bud formation but not thereafter, whereas
CnOtx is also strongly expressed during these early stages but weaker
during later stages. However, neither
gene is expressed during head or
foot regeneration. Because otx
genes are involved in early stages of
anterior patterning in Drosophila as
well as in vertebrate embryos (Finkelstein and Boncinelli, 1994), it is plausible that CnOtx plays a similar role in
the early stages of bud formation.
The initial evagination during bud
formation will form the head of a
hydra. Thus, these two hydra genes,
which are probably involved in the
initiation of the formation of the oral–
aboral axis formation provide molecular evidence that the regeneration of a head involves rebuilding on
an existing axis but not the initiation
of a new axis.
Signalling Molecules Affecting
Head Regeneration
Signalling molecules that play roles
in developmental processes in bilaterians are commonly proteins that
are members of, for example, the
Wnt, TGF, or FGF families. Most likely,
such proteins also play a role in Hydra development, although only a
few have been identified to date.
The Hydra HyWnt gene is the only
one whose function has been inves-
tigated (Hobmayer et al., 2000). In
contrast, several small peptides
(10 –25 amino acids) have been isolated and characterized that affect
axial patterning and regeneration.
Efforts to identify individual signalling
molecules have yielded two that affect head formation, the head activator (Schaller, 1973) and HEADY
(Lohmann and Bosch, 2000), and
two that affect foot formation, pedin and pedibin (Hoffmeister, 1996).
More recently, a large scale effort to
isolate small peptides (⬍30 amino
acids) has yielded approximately
286 purified peptides of which 95
have been sequenced revealing a
large number of novel small peptides (Takahashi et al., 1997; see also
the article by Fujisawa in this issue).
To date, two of these, Hym-346,
which is the same as pedibin, and
Hym-323 have been shown to affect
foot regeneration (Grens et al., 1999;
Harafuji et al., 2001).
The head activator, an 11 amino
acid peptide, was the first of these
peptides to be isolated and characterized (Schaller, 1973; Schaller and
Bodenmuller, 1981). Extensive analysis
indicates that it has several functions.
It stimulates cell cycle traverse
(Schaller, 1976) as well as specifying
interstitial cells for neuron differentiation (Holstein et al., 1986; Hoffmeister
and Schaller, 1987). It also affects
three different aspects of head formation. (1) The head activator stimulates head formation. When applied
to decapitated animals, it increased
the rate of head regeneration as
measured in terms of tentacle formation (Schaller, 1973). In a second
experiment, whole animals were
treated with the peptide, then dissociated, and aggregates formed.
Peptide-treated aggregates were attached to control aggregates. Tentacles and whole heads formed on the
peptide-treated aggregates but not
on the control aggregates (Schaller,
1975). Furthermore, when pieces of
body column that were treated with
the peptide were grafted to untreated pieces, tentacles and head
structures invariably formed on the
head activator-treated pieces but
not on the untreated pieces (Schaller
et al., 1990). These latter two experiments indicate that the peptide has
the capacity to stimulate head for-
mation, including the formation of the
head inhibitor. (2) At a cellular level,
when animals were treated with the
head activator and then decapitated, head-specific epithelial cells
appeared sooner and in greater
number in treated animals than in
controls (Schaller et al., 1990). The
head specificity was measured with
CP8, a head-specific monoclonal antibody (Javois et al., 1986). Similar results were obtained by Hobmayer et
al. (1990, 1997), who showed that the
peptide may more directly affect the
differentiation of body column epithelial cells in the upper part of the
body column into the specific epithelial cells of the tentacles. (3) The third
effect of the peptide is to stimulate
bud formation (Hobmayer et al.,
1997). That is, buds appear earlier
than they would in untreated controls.
This role may be similar to the one
mentioned above in which the peptide stimulates head formation.
More recently Lohmann and
Bosch (2000) have isolated and analyzed the role of a gene, Heady,
that encodes a 23 amino acid peptide. The gene is strongly expressed
early during head regeneration as
well as in the apical regions of developing buds. However, no expression was detected in adults, suggesting the peptide has a role in head
formation but not head maintenance. More direct evidence for its
role in head regeneration was obtained by using RNAi. Heady dsRNA
was introduced into whole Hydra
with electroporation, and they were
subsequently decapitated. Head regeneration was clearly delayed in
those treated with dsRNA. Additional evidence was obtained by
treating whole animals with the peptide and carrying out transplantation experiments, which indicated
that the transplant had a higher capacity for head formation than did
untreated control transplants. It is
unclear where in the process of
head formation this peptide is active.
Morphogenesis During Head
Regeneration
After the two regions of the head,
the hypostome and tentacle zone,
have been specified, the next de-
CHARACTERIZATION OF THE Hydra ORGANIZER 233
velopmental events involve their
morphogenesis. As described previously (see Fig. 3), after decapitation,
the open wound is sealed by the
stretching of the ectoderm and
endoderm over the wound to form
an apical cap. Approximately 30 –36
hr after decapitation, tentacle buds
begin to emerge in the tentacle
zone, which will subsequently elongate into tentacles as tissue from the
body column is displaced apically
through the tentacle zone onto the
growing tentacles. The second
change is more subtle and involves
the flat to slightly rounded apical
cap changing its shape into a more
dome-like or conical shape as it develops into the hypostome.
As epithelial tissue is displaced
from the tentacle zone and onto the
tentacles, the epithelial cells of both
layers undergo drastic changes in
shape (see Fig. 1). In the body column and the tentacle zone, these
cells have cylindrical shapes with
one end of each cylinder attached
to the underlying basement membrane between the two epithelial
layers. As they move from the tentacle zone onto the tentacles, these
epithelial cells shift to a much flatter
shape with a much larger surface
area now in contact with the mesoglea. This change is especially pronounced in the endoderm. The flattening of the epithelial cells along
the tentacle starts sharply near the
tentacle zone/tentacle border but
continues through a considerable
part of a tentacle. This would require
an increase in the area and, thus,
the amount of basement membrane available. At the same time,
the area of contact between two
neighboring epithelial cells would be
drastically reduced.
Correspondingly, one would expect the expression of several genes
involved in the production of proteins that make up the basement
membrane to be stronger in the
area of the tentacle zone/tentacle
(TZ/T) border as well as further along
on the tentacles. Sarras and colleagues have been systematically
isolating genes from Hydra that are
commonly involved in the construction of the basement membrane in
bilaterians, and characterized their
roles. Hydra genes encoding base-
ment membrane components, i.e.,
type IV collagen, a metalloproteinase 1 (HMP1), the beta-1 chain of
laminin (HLM-␤1) and HMMP, which
is a Hydra matrix metalloproteinase,
are strongly expressed around the
TZ/T border and into the tentacles
(Fowler et al., 2000; Yan et al., 2000;
Shimizu et al., 2002; Leontovich et al.,
2000). In addition, a Hydra IQGAP
gene is also strongly expressed in this
region (Venturelli et al., 2000). In
mammals, this protein induces the
loss of E-cadherin–mediated cell–
cell adhesion. In Hydra, expression of
IQGAP could lead to the reduction
of cell– cell contact between neighboring epithelial cells, leading to a
change in cell shape from cylindrical to squamous, or flat.
Decapitation of a Hydra results in
the retraction of the mesoglea, the
basement membrane between the
two epithelial layers, from the site of
the wound. Subsequently, during
head regeneration, the mesoglea is
rebuilt by synthesis of mesogleal
components (Shimizu et al., 2002).
Correspondingly, there is an up-regulation of genes encoding mesogleal components in the apical
cap initially and later during tentacle formation. This is true of (1)
Hcol-1, a fibrillar collagen; (2) type IV
collagen; (3) HMP1; (4) HLM-␤1, and
(5) HMMP (Deutzmann et al., 2000;
Fowler et al., 2000; Yan et al., 2000;
Shimizu et al., 2002; Leontovich et al.,
2000). Hcol-1 and HLM-␤1 were upregulated within 3 hr and continued
at this high level for 4 days (Shimizu
et al., 2002). A high level of expression is found initially all over the apical cap and then shifts by 30 to 48 hr
to the emerging tentacle buds and,
subsequently, in the adult pattern of
the TZ/T region and the tentacles.
That this rebuilding of the mesoglea
is required for head regeneration
was shown for this set of genes by
introducing an antisense oligonucleotide into tissue followed by decapitation for each of four of the five
genes and an antibody for HMP1. In
each case, head regeneration was
inhibited for several days (Deutzmann et al., 2000; Fowler et al., 2000;
Shimizu et al., 2002). Thereafter,
head regeneration took place as
presumably the antisense oligonucleotides were digested. Finally, as
epithelial cells involved in tentacle
formation during head regeneration
will change from a cylindrical to a
squamous shape, one would also
expect IQGAP to be up-regulated,
and it is (Venturelli et al., 2000).
The morphogenetic changes during hypostome formation are more
subtle. During regeneration, the apical cap, which will form the hypostome changes shape from a flat
cap to the dome-like or conical
shape of the adult hypostome (Fig.
3). The formation of the dome/cone
will result in rings of cells with progressively smaller diameters and small
numbers of cells within a ring as the
dome/cone emerges from the cap.
Subsequently, this structure is maintained in the context of the tissue
movements in the hypostome. Because the diameter of the dome decreases, the number of cells per ring
must decrease when moving in an
apical direction. This decrease is
most likely accomplished by cells
continuously shuffling past one another to reduce the number of cells
per ring as the tissue is displaced in
an apical direction. During bud formation, a similar phenomenon occurs. Bud formation begins with the
evagination of part of the body column wall, which is subsequently converted into a cylindrical protrusion.
The changes in location of neighboring ectodermal cells was observed
by marking a patch of contiguous
cells just as evagination began. By
the time the bud had developed
into a cylindrical protrusion, the
roughly circular patch had changed
into an elongated cylinder or line of
marked cells clearly indicating that
cells had changed position with respect to one another (Smith et al.,
1999).
The behavior of the epithelial tissue in the steady state of the hypostome as well as bud formation is very
similar to convergent extension of tissue observed during gastrulation in
bilaterians. In both Drosophila and
Xenopus, the blastopore, the part of
the embryonic wall that will form the
hindgut, invaginates and subsequently changes shape from the
shallow cylinder with a large diameter to that of a long slender cylinder.
This change involves convergent extension.
234 BODE
Because a similar set of genes is
expressed in Drosophila and Xenopus as well as several other organisms during this early phase of gastrulation, Lengyel and Iwaki (2002)
have suggested that an evolutionarily conserved “cassette” of genes
is involved in the mechanism underlying convergence and extension.
The common set of genes expressed
during early stages of gastrulation in
each organism examined contains
Wnt, Brachyury, frk/HNF-3␤, and cdx.
Hydra homologues of three of these
genes, HyWnt, HyBra1, and budhead, which is a HNF-3␤ homologue,
are expressed in the hypostome (Fig.
5) as well as in the evaginating tissue
of the bud (Martinez et al., 1997;
Technau and Bode, 1999; Hobmayer
et al., 2000). A Hydra cdx has not
been identified yet. As all three
genes are expressed before the apical cap is converted into a dome
during head regeneration as well as
during the initial evagination of a
bud, it is plausible that they are acting as part of the “cassette” to convert the cap into a dome during
head regeneration. In this case, instead of dealing with an invagination as during gastrulation, the cassette may be controlling a pair of
evaginations. Because two of the
genes, HyWnt and HyBra1, are implicated in patterning of the hypostome, these genes may have at
least two roles during head regeneration as well as in the maintenance
of the hypostome in the steady state
adult.
PERSPECTIVES
The processes underlying regeneration of a structure, be it epimorphic
or morphallactic, are most likely the
same, or quite similar, to the processes leading to the formation of
the structure during embryogenesis.
In Hydra, little is known about the
molecular events governing embryogenesis. However, a set of patterning and morphogenetic processes
are constantly active in the context
of the tissue dynamics of the adult
animal to maintain its morphology.
As shown so far, the patterning processes governing head regeneration involve rebuilding the head organizer and the two gradients,
which are the central ingredients of
the patterning processes for maintaining the steady state of the adult
animal. To gain a more detailed understanding of the relationship between these processes in the steady
state and head regeneration will require an increased understanding of
the molecular mechanisms involved
in both sets of processes. In particular, the application of DNA arrays
may lead to a more rapid identification of the genes involved in maintenance of the head as well as during
head regeneration.
A closely related issue involves the
relationship between the regeneration of a head and the formation of
a new head, which occurs during
bud formation. The latter involves
the initiation of a new axis, whereas
head regeneration clearly involves
simply repairing an existing axis. The
identification of two transcription
factors, Cnotx (Smith et al., 1999)
and Hymsx (McCord and Bode,
manuscript in preparation), that are
expressed only during bud formation
and not during head regeneration,
indicate that there will be differences at a molecular level. Identifying more genes involved in either
only bud formation or head regeneration will help elucidate the differences between the two processes.
Another aspect is the topic of foot
regeneration. As the emphasis in the
field has been on the patterning and
regeneration of the head, less is
known about foot formation and regeneration. Currently, the relationship between foot formation and
the morphogenetic gradient is unclear. Furthermore, there is some evidence for an interaction between
the head and foot ends during regeneration of the foot (e.g., Muller,
1990; Grens et al., 1996), but these
effects need to be clarified. At a
molecular level, efforts are now being made to uncover the genes involved in foot formation, which include the signalling molecules
described above as well as transcription factors and receptors (e.g.,
Bridge et al., 2000).
Finally, another subject of interest
are the signalling molecules. The
finding of several small peptides that
affect patterning processes in hydra
could lead to the view that this ani-
mal, and plausibly other primitive
metazoans, makes more use of such
molecules for signalling purposes in
developmental contexts than do bilaterians. This could be the case,
although the sets of signalling molecules are not exclusive. The presence of a Wnt gene indicates the
use of a common signalling molecule in a central developmental
process in Hydra. Evidence for the
presence of Hedgehog (Kaloulis,
2000) and components of a TGF-␤
signalling pathway (BMP5-7 [Reinhardt and Bode, unpublished results]; chordin [Hobmayer, Rentsch,
and Holstein, personal communication) indicates that several signalling
pathways found in bilaterians exist in
Hydra and no doubt other diplobasts. Conversely, Schaller and colleagues have shown that the head
activator exists in mammals and affects neuron differentiation (Schaller
et al., 1989). Identification and characterization of more of the signalling
molecules should shed some light on
this issue as well as provide information on the evolution of signalling
molecules in developmental processes.
ACKNOWLEDGMENT
I thank the National Science Foundation for their generous support of
the research in my laboratory during
recent years.
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