Department of Developmental and Cell Biology and the Developmental Biology Center, University of California, Irvine, CA 92697, USA
* Author for correspondence (e-mail: hrbode{at}uci.edu)
Accepted 4 April 2005
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SUMMARY |
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Key words: Hydra, Head organizer, Canonical Wnt pathway
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Introduction |
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Among the diploblasts, these processes have been extensively investigated
in the cnidarian hydra. The hydra polyp consists of a single axis with radial
symmetry. The regions along the axis are the head, body column and foot.
Because of the tissue dynamics of an adult hydra, the processes governing
axial patterning are continuously active (e.g.
Bode, 2003). Similarly, as bud
formation, a form of asexual reproduction, also occurs continuously in hydra,
the processes governing the initiation of axis formation are also constantly
active. Axial patterning, especially of the head, is well understood in hydra
at the tissue level. The head organizer
(Browne, 1909
;
Technau et al., 2000
;
Broun and Bode, 2002
) located
in the hypostome, the upper portion of the head in a hydra, produces a signal
and transmits it to the body column. The signal sets up a morphogenetic
gradient that decreases down the body column. This gradient, referred to as a
positional value gradient (Wolpert,
1971
), a source density gradient
(Gierer and Meinhardt, 1972
)
or the head activation gradient
(MacWilliams, 1983b
), confers
head formation capacity on tissue of the body column. The head organizer also
produces a second signal, head inhibition, which is transmitted to and graded
down the body column. Head inhibition prevents body column tissue from forming
heads (MacWilliams,
1983a
).
The molecular basis of the head organizer and these gradients is not well
understood. Homologs of a number of genes that affect axial patterning in
bilaterians have been isolated from hydra, and appear to play similar roles.
For example, the expression patterns of Cnox-3, a homolog of the Hox
gene labial/Hox-1, and of the parahox gene Cnox-2, a homolog
of Gsx, in several experimental situations suggest that these two
genes are involved in the control of head formation
(Shenk et al., 1993a;
Shenk et al., 1993b
;
Bode, 2001
).
With respect to the head organizer, genes of the canonical Wnt pathway have
been identified in hydra, and their expression patterns suggest that this
pathway plays a role in this structure
(Hobmayer et al., 2000).
HyWnt is expressed exclusively in the hypostome, where the head
organizer is located. The gene is also expressed very early in the apical tip
of the developing head during head regeneration and bud formation as the head
organizer is developing. HyTcf has a similar, although slightly more
extended, range of expression, and Hyß-Cat is expressed
strongly during early stages of head formation.
In addition, there are data implicating glycogen synthase kinase-3ß in
the activity of the head organizer. Treatment with LiCl, which inhibits
GSK-3ß (e.g. Phiel and Klein,
2001), results in the formation of ectopic tentacles along the
body column (Hassel et al.,
1993
). Exposure to diacylglycerol, which activates protein
kinase-C (PKC) (Nishizuka,
1992
), which in turn blocks GSK-3ß activity
(Goode et al., 1992
), causes
the formation of individual ectopic tentacles or complete heads along the body
column (Mueller, 1989
).
However, both reagents have other effects. For example, Li+ blocks,
and diacylglyercol catalyzes, the traverse of the phosphoinositol pathway
(Hallcher and Sherman,
1980
).
To gain more direct evidence for the role of the canonical Wnt pathway in
the formation of the head organizer, we made use of alsterpaullone, which
specifically blocks the activity of GSK-3ß
(Leost et al., 2000;
Bain et al., 2003
). Treatment
with this inhibitor blocked GSK-3ß activity throughout the animal as well
as elevating the level of ß-catenin in the nuclei of body column cells.
The treatment also conferred characteristics of the head organizer on the body
column as well as inducing the expression of genes of the Wnt pathway in the
body column. These results provide direct evidence for the role of the
canonical Wnt pathway in the formation and maintenance of the head organizer
in hydra.
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Materials and methods |
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Tissue manipulations
Treatment with alsterpaullone
Hydra were exposed to 5 µmol/l alsterpaullone (A.G. Scientific, Inc.) in
0.025% DMSO in hydra medium for 1 or 2 days. Thereafter, they were rinsed
several times and cultured in hydra medium for 25 days. Treatment with
0.025% DMSO had no effect on hydra. To determine if treatment with
alsterpaullone affected cell cycle traverse, 1 µmol/l BrdU was injected
after 2 days of treatment and the labeling index was measured, as described by
Bode et al. (Bode et al.,
1990).
Treatment with aminopurvalanol
Hydra were exposed to several concentrations of aminopurvalanol for 2 days,
and then returned to hydra medium without inhibitor. The highest concentration
of the reagent used, 20 µmol/l, resulted in a final concentration of 0.2%
DMSO, which had no effect on hydra.
Transplantation experiments
To assay the inductive capacity of body column tissue treated with
alsterpaullone, one-eighth of the body column of a donor animal was isolated,
cut into four equal-sized pieces, and one of these quarters grafted into the
body column of a host animal, as described previously
(Broun and Bode, 2002). Host
animals were injected with India ink
(Campbell, 1973
) before
transplantation to distinguish transplanted from host tissue.
Experiments to measure the capacity of body column tissue to produce either
head inhibition or the signal for setting up the head activation gradient were
carried out as described by Wilby and Webster
(Wilby and Webster, 1970a;
Wilby and Webster, 1970b
).
Donor animals were labeled with India ink, and then treated with
alsterpaullone for 2 days. Thereafter, one-eighth of the length of the body
column of the treated animal was isolated and grafted onto the basal end of a
bisected host animal, as described by Rubin and Bode
(Rubin and Bode, 1982
).
Isolation of GSK-3ß
GSK-3ß was isolated from hydra by affinity chromatography using axin
sepharose beads, as described by Primot et al.
(Primot et al., 2000). Two
adult hydra were homogenized in 100 µl of a buffer, which was a combination
of the homogenization buffer and the lysis buffer used for cell culture
(Primot et al., 2000
). This
combination buffer consisted of the components of the lysis buffer plus 60
mmol/l ß-glycerophosphate, 50 mmol/l Na vanadate, 500 mmol/l NaF, 10
µg/ml leupeptin, 10 µg/ml aprotenin, 10 µg/ml SBTI and 100 µmol/l
benzamidine. The lysate was centrifuged at 14,000 g in a
microfuge for 5 minutes at 4°C. Subsequently, the supernatant was mixed by
rotation for 30 minutes at 4°C with 5 µl axin-sepharose beads in 25
µl bead buffer (beads were stored as a 20% suspension in a bead buffer)
plus another 25 µl of bead buffer. Then the beads were washed 3 x
with 100 µl of bead buffer, 2 x with 100 µl kinase buffer and,
finally, resuspended in 50 µl kinase buffer as described by Ryves et al.
(Ryves et al., 1998
).
GSK-3ß kinase assay
GSK-3ß kinase activity was measured using 32P-ATP as
described by Ryves et al. (Ryves et al.,
1998) using the GSM [RRRPASVPPSPSLSRHSSHQRR] peptide, in which the
underlined serine is phosphorylated. GSM(nP), which is the same peptide
without the phosphorylated serine, served as a control. Three microliters of
either hydra lysate or GSK-3ß purified on axin-sepharose beads was used
in a 12 ml reaction mixture. To assay the inhibitory effect of alsterpaullone
on GSK-3ß, the kinase assay was carried out on axinsepharose-isolated
GSK-3ß in the presence of different concentrations of alsterpaullone.
Immunocytochemistry on whole mounts
Staining with the TS-19 monoclonal antibody was carried out as described by
Bode et al. (Bode et al.,
1988). For the anti-ß-catenin antibody the following
procedure was used. Animals were relaxed in 2% urethane for 2 minutes, and
then fixed in Lavdowsky's fixative (50% ethanol, 10% formalin, 4% acetic acid,
40% water) for 10-15 minutes. Then the animals were washed 3 x 5 minutes
in PBS, and once for 5 minutes by rotation with PBST. Next, the animals were
incubated with rotation in 4% goat serum in PBST for 60 minutes at 4°C.
After this, they were incubated overnight with a 1:100 dilution of an
affinity-purified guinea pig anti-sea urchin ß-catenin polyclonal
antibody (Miller and McClay,
1997
). After washing 4-5 times for 5 minutes with 4% serum,
samples were incubated for 60 minutes at room temperature with a 1:100
dilution of a FITC-conjugated donkey anti-guinea pig polyclonal antibody
(Jackson ImmunoResearch Laboratories). Subsequently, the sample was incubated
in a 1:100 dilution of propidium iodide for 20-30 minutes at room temperature.
Finally, the animals were washed 2 x 10 minutes in PBST, and then 2
x 10 minutes in PBS. They were mounted using Vectashield mounting medium
(Vector Laboratories, Inc.) and stored at 80°C. Samples were
examined with confocal microscopy.
In situ hybridization
In situ hybridization analysis was carried out on whole mounts of hydra as
described previously (Grens et al.,
1996; Martinez et al.,
1997
). The antisense RNA probe for HyBra1 was a fragment
of the gene containing the 5' end of the ORF including the T-box domain.
For the HyWnt and HyTcf genes, the antisense RNA probes contained the
entire open reading frame. Probes were used at concentrations 0.025 ng/µl
for Hybra1, 0.1 ng/µl for HyWnt and 0.01 ng/µl for
HyTcf.
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Results |
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Alsterpaullone also blocks three cyclin-dependent kinases, CDK 1, 2 and 5
(Leost et al., 2000). The fact
that the observed effects of alsterpaullone on the formation of head
structures was not due to inhibition of these enzymes was shown as follows.
Aminopurvalanol is a specific inhibitor of CDKs
(Leost et al., 2000
) but has
no effect on GSK-3ß (Bain et al.,
2003
). Treatment of hydra with several concentrations of
aminopurvalanol (1, 2 or 5 µmol/l) for 2 days did not induce the formation
of ectopic head structures, nor did it have any other effects on the
morphology of the animal. Treatment with 10 or 20 µmol/l aminopurvalanol
resulted in the disintegration of hydra within 1 day, indicating that the
inhibitor penetrated the tissues of the animal (data not shown).
Another assay involved the roles of CDK1 and CDK2 in cell cycle traverse.
If alsterpaullone was blocking these enzymes one would expect a reduction in
the fraction of cells in S-phase, as the two enzymes are involved in the G1/S
and G2/M transitions (Murray,
2004). Animals were treated with 5 µmol/l alsterpaullone for 2
days and then injected with 1 µmol/l BrdU. The labeling index of the
epithelial cells and the interstitial cells did not decrease
(Table 1), indicating that
neither of the two enzymes was inhibited by alsterpaullone. Instead, the
labeling index increased, which is consistent with another effect of blocking
GSK-3ß activity. CDK4, which is essentially not affected by
alsterpaullone (Leost et al.,
2000
), also affects cell cycle progression. Phosphorylation of
CDK4 by GSK-3ß leads to its degradation and, hence, slows down cell cycle
traverse (Ewen et al., 1993
).
Blocking GSK-3ß with alsterpaullone increases the level of CDK4 activity,
thus, enhancing cell cycle traverse, which could account for the observed
increase in the labeling index.
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Inhibition of GSK-3ß increases the level of ß-catenin in the nucleus and cell membranes of hydra
In the canonical Wnt pathway, GSK-3ß phosphorylates ß-catenin,
targeting it for degradation (Yost et al.,
1996). Blocking the activity of GSK-3ß results in the
accumulation of ß-catenin in cell membranes, where the protein has a role
in cell-cell adhesion (Nagafiuchi and
Takeichi, 1989
). Accumulation of ß-catenin also occurs in the
nuclei of cells, where, along with Tcf, it is involved in transcription of
genes downstream of the Wnt pathway
(Behrens et al., 1996
). To
determine if blocking GSK-3ß activity by treatment with alsterpaullone
had similar effects on ß-catenin in hydra, we made use of an
affinity-purified guinea pig anti-sea urchin ß-catenin polyclonal
antibody (Miller and McClay,
1997
). This antibody is known to bind to the ß-catenin of
tunicates and amphioxus as well as sea urchins (D. McClay, personal
communication). Three different circumstances were analyzed.
Hydra treated with 5 µmol/l alsterpaullone for 24 hours were subjected to immunocytochemistry with anti-ß-catenin antibody and examined with confocal microscopy. The treatment raised the level of ß-catenin associated with the cell membranes (Fig. 3B) compared with controls (Fig. 3A).
HyWnt, the hydra Wnt homolog, is expressed exclusively in
the apical tip of the hypostome of an adult hydra
(Hobmayer et al., 2000). If
GSK-3ß is a negative regulator of ß-catenin downstream of
Wnt in hydra as it is in other systems, one would expect higher
levels of nuclear ß-catenin in the hypostome than elsewhere in the adult.
This was confirmed by examining the head, body column and foot regions of an
adult. As shown in Fig. 4A,B,
many of the nuclei in the apical tip of the hypostome were strongly labeled
with the antibody. The smaller nuclei are the nuclei of neurons, while the
larger nuclei are those of epithelial cells. This was observed nowhere else in
the adult, for example in the body column
(Fig. 4C,D).
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Treatment with alsterpaullone confers characteristics of the hypostome on the body column
The increased level of nuclear ß-catenin in the body column of animals
treated with alsterpaullone suggests the body column may have taken on
characteristics of the hypostome. To examine this possibility, the expression
patterns of three genes expressed exclusively or predominantly in the
hypostome were observed.
HyBra1, a hydra brachyury homolog, is expressed in the
hypostome of an adult hydra (Fig.
6A) (Technau and Bode,
1999). Treatment with 5 µmol/l alsterpaullone for 24 hours
expanded the expression domain of the gene throughout most of the body column
(Fig. 6B).
The two other genes examined, HyWnt and HyTcf, the hydra
homologs of Wnt and Tcf, encode proteins that are part of
the canonical Wnt pathway. The change in the expression pattern of
HyTcf in response to alsterpaullone was similar to that of
HyBra1. Normally HyTcf is expressed in the head region of an
adult (Fig. 6C), with the most
intense level of expression in the apex of the hypostome
(Hobmayer et al., 2000). After
treatment with alsterpaullone for 1 day, HyTcf was expressed
throughout the body column (Fig.
6D). HyWnt is expressed exclusively in the apical tip of
the hypostome (Hobmayer et al.,
2000
). After 24 hours of treatment the HyWnt expression
domain expanded from the apical tip (Fig.
6E) to cover a larger area of the hypostome (data not shown).
After 48 hours of treatment, spots of HyWnt expression similar in
size to the spot in the apical tip of the hypostome appeared in the upper
two-thirds of the body column in a relatively regularly spaced pattern
(Fig. 6F).
Thus, treatment with alsterpaullone, which inhibited GSK-3ß activity, induced ectopic expression of the head-specific genes, Hybra1, HyWnt and HyTcf, and subsequently the formation of tentacles and ectopic heads along the body column.
Alsterpaullone promotes formation of the head organizer in the body column
As the expression of HyWnt is associated with the head organizer,
the expression of the gene in the body column suggests the body columns of
alsterpaullone-treated animals may have acquired characteristics of the head
organizer. As described in the Introduction, the head organizer is defined by
three properties: (1) its ability to induce a second axis when transplanted to
the body column of a host animal; (2) the production and transmission of a
signal that sets up the head activation gradient; and (3) the production and
transmission of a head inhibition signal that prevents head formation from
occurring in the body column. These three properties were examined to
determine if treatment with alsterpaullone had conferred head organizer
characteristics on tissue of the body column.
Capacity for induction of a second axis
When a hypostome is transplanted into a body column, it invariably induces
body column tissue to form a second axis consisting of a head and body column
(Browne, 1909;
Broun and Bode, 2002
). As a
similar-sized piece of tissue (1/32nd) taken from any part of the body column
does not form a second axis upon transplantation
(Yao, 1945
;
Broun and Bode, 2002
), this
inductive capacity is restricted to the hypostome.
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None of the control transplants, that is, a quarter of the 3-region of a
control animal, formed a second axis (Fig.
7C). By contrast, the same piece of tissue from an animal treated
for 1 day with alsterpaullone formed a second axis in 40% of the
transplants, while a 2-day treatment resulted in all of the transplants
forming a second axis (Fig.
7C). The fact that the second axis was due to induction is shown
in Fig. 7B, in which a quarter
of a 3-region of an alsterpaullone-treated animal was transplanted into a host
animal stained with India ink. The hypostome of the second axis is derived
from the unstained transplant, while the rest of the second axis (tentacles
and body column) is made up of host tissue.
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If instead of a head, a 4-region of a normal animal was grafted to the basal end of the H-1-2-3-4 region (2-4/4) the result was similar to the one in which the 2-4 region is allowed to regenerate. Most of the samples regenerated a head at the 2-end (Fig. 8B, 2-4/H), indicating that the grafted 4-region has little or no capacity to inhibit head regeneration. By contrast, when a 4-region from a donor treated with 5 µmol/l alsterpaullone for 2 days was grafted to the basal end (2-4/ALP-4), and the same experiment was carried out, none of the samples regenerated a head (Fig. 8B, 2-4/ALP-4). This indicates that the alsterpaullone-treated 4 region has the same capacity as a head to produce and transmit head inhibition along the body column to prevent head regeneration at the apical end.
Capacity to produce and transmit a signal that sets up the head activation gradient
The head activation gradient is maximal at the apical end of the body
column and declines along its length (e.g.
MacWilliams, 1983b). This
gradient confers regeneration polarity on the body column. That is, when a
piece of the body column is isolated, a head invariably regenerates at the
apical end. Wilby and Webster (Wilby and
Webster, 1970b
) demonstrated that grafting a head to the basal end
of a piece of the body column will invert this regeneration polarity. When the
grafted head is removed, a head will regenerate at the basal end due to this
inversion. This experiment provided direct evidence that the head produces a
signal that sets up the head activation gradient.
A similar experiment was carried out using the four conditions described in the previous experiment. An isolated 2-4 region regenerated a head at the apical or 2-end (Fig. 9B). When a head was grafted to the 4-end (2-4/H), the host head removed after 6 hours, and then the remaining graft left intact for 6 days before removing the grafted head (Fig. 9A), a different result was obtained. Half the grafts regenerated a head at the basal end, while very few regenerated a head at the apical end (Fig. 9B, 2-4/H), which indicates that the head activation gradient had been inverted in many of the animals. Variations of this graft were carried out using a 4-region from an alsterpaullone-treated animal (2-4/ALP-4), and a control 4-region (2-4/4). Invariably, a head regenerated at the apical end when a control 4-region was used (Fig. 9B, 2-4/H). By contrast, the alsterpaullone-treated 4-region behaved like a head, in that in a similar number of samples the head activation gradient was inverted (Fig. 9B, 2-4/ALP-4). Hence, treatment with alsterpaullone confers the characteristic of the head organizer on the body column to send out a signal setting up the head activation gradient.
Thus, these three experiments indicate that alsterpaullone treatment confers characteristics of the head organizer on the body column.
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Discussion |
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A crucial step in the canonical Wnt pathway is the stabilization and
subsequent accumulation of ß-catenin in the nucleus due to the inhibition
of GSK-3ß activity. By blocking the activity of GSK-3ß, the
formation of an ectopic organizer or ectopic axial structures can be induced
in a number of bilaterians (Dominguez et
al., 1995; Diaz-Benjumea and
Cohen, 1994
; Sumoy et al.,
1999
). We show here that a similar phenomenon occurs in hydra.
The Wnt pathway is involved in the head organizer in hydra
Three sets of results indicate that the Wnt pathway plays a central role in
the formation and maintenance of the head organizer in hydra. One is the set
of expression patterns of three genes, HyWnt, HyTcf and
Hyß-Cat, of the canonical Wnt pathway. They are
expressed in the mature hypostome or in a developing hypostome
(Hobmayer et al., 2000),
suggesting that this pathway plays a role in the head organizer.
Transplantation experiments indicate that blocking GSK leads to HO formation
The second set involves the transplantation experiments, which demonstrate
that blocking GSK-3ß activity throughout the animal leads to head
organizer formation in the body column. When hydra were treated with
alsterpaullone, it clearly blocked the activity of GSK-3ß but had no
inhibitory effect on the CDKs. Blocking GSK-3ß activity resulted in
tissue of the body column, acquiring the three known characteristics of a head
organizer.
The fundamental property of the head organizer in hydra is its ability to
induce a second axis when the hypostome is transplanted to the body column
(Broun and Bode, 2002). A piece
of normal body column tissue similar in size to that of a hypostome does not
have this ability (Yao, 1945
;
Broun and Bode, 2002
). By
contrast, a hypostome-sized piece of alsterpaullone-treated body column tissue
does have this inductive capacity, and has acquired a level similar to that
found in a normal head.
The other two properties involve the two signals produced by the head organizer and transmitted down the body column, which set up the head activation and head inhibition gradients. Normal body column tissue does not generate either of these two signals. However, the tissue of an alsterpaullone-treated body column produces both signals. A piece of such a body column can invert the head activation gradient when grafted to the basal end of a body column, indicating that it is producing the signal for setting up this gradient. And, at the same time, this piece of tissue produces head inhibition and transmits it up the body column. The produced and transmitted level of head inhibition is sufficiently high to prevent head regeneration from taking place at a considerable distance (one-third to half the body length) from the source of this head inhibition, the alsterpaullone-treated piece of body column.
Thus, blocking the activity of GSK-3ß conferred all three characteristics that define a head organizer onto tissue of the body column.
Blocking GSK-3ß results in head genes expressed in the body column
As inhibition of GSK-3ß confers head organizer characteristics on the
body column, one would expect genes expressed in the hypostome or associated
with the head organizer, such as genes of the canonical Wnt pathway, to be
expressed in the body column. Normally, HyBra1, the hydra homolog of
Brachyury, and HyWnt are expressed only in the head, while
HyTcf is expressed much more strongly in the head than in the body
column (Technau and Bode,
1999; Hobmayer et al.,
2000
). In addition, staining with an anti-ß-catenin antibody
indicates that the nuclei of the hypostome contain high levels of the
ß-catenin protein, while levels in the body column are much lower.
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In sum, the latter two sets of data provide more direct evidence for the involvement of the Wnt pathway in the formation of the head organizer. Blocking the activity of GSK-3ß results in the body column acquiring the characteristics of the head organizer as well as expressing genes associated with the Wnt pathway.
The pattern of HyWnt expression following GSK-3ß inhibition reflects the patterning processes governing head formation
Another result is consistent with the Wnt pathway playing a role in the
head organizer. Instead of HyWnt being uniformly expressed throughout
the body column of alsterpaullone-treated animals, it was expressed in spots
that were similar in size to the spot of its expression found in the
hypostome. This spotted pattern can be explained in terms of the head
organizer and head inhibition.
In the normal animal, the organizer in the head produces head inhibition,
which prevents the formation of new head organizers nearby. Only further down
the body column where the head inhibition level falls below a threshold value,
will a new head organizer arise. This results in the formation of the axis of
a new bud, hydra's asexual form of reproduction. HyWnt is expressed
as a spot on the body column, where this next bud will form, and continues to
be expressed in the apical tip of the developing bud
(Hobmayer et al., 2000).
As blocking GSK-3ß activity led to a uniform rise in the level of HyTcf transcription throughout the body column, one might expect a similar rise in HyWnt expression. As it is likely that the rise probably occurs unevenly, high levels of HyWnt will initially appear in random spots. And, if the rise in HyWnt is responsible for, or directly coupled with head organizer formation, then these random spots will form head organizers and begin producing head inhibition. In turn, these organizers will prevent the formation of other head organizers in their immediate vicinity. This could lead to the observed fairly uniform distribution of HyWnt-expressing spots, each of which is a putative head organizer region. Each of the spots is similar in size to the one in the hypostome, which is also consistent with each of them being associated with a head organizer.
As these head organizers in the body column continuously produce head inhibition, eventually one would expect rising levels of head inhibition throughout the body column. This was observed in the head inhibition experiment (Fig. 8). The amount of head inhibition produced by a piece of body column tissue derived from an alsterpaullone-treated animal was similar to, if not more than, that produced by a normal head. Plausibly, this reflects the presence of more than one head organizer in the piece of body column used in the transplant, as indicated by the presence of more than one HyWnt spot in this piece.
The elevated levels of head inhibition, coupled with an elevated level of head activation in the body column, also provide an explanation for the formation of ectopic tentacles but no complete heads in alsterpaullone-treated animals. In a normal animal, the head organizer in the hypostome produces and transmits head inhibition down the body column, preventing tissue of the body column from initiating head organizer, and hence head, formation. In the alsterpaullone-treated animals, the level of head inhibition is so high due to the multiple head organizers, that complete head formation involving hypostome and tentacle zone does not take place.
By contrast, tentacle formation is controlled by the head activation
gradient, and is not affected by head inhibition. The head organizer produces
and transmits a signal to the body column that sets up the head activation
gradient in the body column in a normal hydra
(MacWilliams, 1983b). Tentacle
formation occurs above a threshold level of head activation. This is reflected
in the commitment of tissue just below and in the tentacle zone to tentacle
formation (Hobmayer et al.,
1990
). In addition, treatment with LiCl raises the head activation
level in the body column (L.G. and H.R.B., unpublished), which leads to the
formation of ectopic tentacles on the body column
(Hassel et al., 1993
). The
multiple organizers in the alsterpaullone-treated animals most probably
produce the signal for head activation, thereby generating a high level of
head activation throughout the body column. In turn, this level is probably
above the threshold level for tentacle formation, resulting in the large
number of ectopic tentacles observed in alsterpaullone-treated animals.
Alsterpaullone also resulted in an elevated level of cell division, as
indicated by an increased labeling index. However, it is unlikely that this
effect is related to the formation of the ectopic tentacles or the head
organizer activity in the body column. The patterning processes in hydra are
morphallactic, and hence independent of cell division. For example, isolation
of a piece of the body column results in the regeneration of a head at the
apical end and a foot at the basal end and the proportions of a normal animal.
The same pattern of regeneration takes place in the presence or absence of
cell division (Cummings and Bode,
1984).
Role of the Wnt pathway in the maintenance of the head organizer
Should the Wnt pathway play a central role in the head organizer, it would
also provide an explanation for the maintenance of the head organizer in the
context of the tissue dynamics of an adult hydra. In an adult hydra, the
tissues are in a steady state of production and loss. Cells of all three cell
lineages in the body column are continuously in the mitotic cycle. These are
the epithelial cells of both the ectoderm and endoderm, as well as the
interstitial cells of the interstitial cell lineage
(David and Campbell, 1972;
Campbell and David, 1974
). To
maintain the size of the animal, tissue is displaced apically into the head
and basally onto the foot from the body column, and eventually sloughed at the
extremities (Campbell, 1967
).
Since the hypostomal tissue is continuously displaced toward its apex and
lost, the head organizer must also be in a steady state of production and
loss.
The canonical Wnt pathway is known to act as a positive feedback loop in
Drosophila (Heslip et al.,
1997). If it acted in a similar manner in hydra, it could be
involved in maintaining the head organizer. The Wnt signal produced by the
head organizer in the tip of the hypostome could stimulate neighboring cells
just basal to the tip by blocking GSK-3ß and raising the nuclear level of
ß-catenin. In addition, HyPKC2, a hydra PKC homolog
that is expressed in the apical half of the hypostome
(Hassel et al., 1998
), may
augment this activity, as PKC is known to inhibit the activity of GSK-3ß
(Goode et al., 1992
).
Subsequently HyWnt would be transcribed in the neighboring cells,
completing the positive feedback loop. Hence, the Wnt positive feedback loop
would be continuously active, which would maintain the head organizer in the
context of the continuous displacement toward, and loss of tissue at, the apex
of the hypostome. This would also be consistent with the continuous expression
of HyWnt and HyTcf in the hypostome
(Hobmayer et al., 2000
).
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ACKNOWLEDGMENTS |
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