1 Philipps University Marburg, FB 17, Morphology and Evolution of Invertebrates,
Karl von Frisch Strasse 8, 35032 Marburg, Germany
2 INSERM Unité 119, 27 boulevard Lei Roure, 13009 Marseille, France
* Author for correspondence (e-mail: hassel{at}staff.uni-marburg.de)
Accepted 6 May 2004
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SUMMARY |
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Key words: FGFR, Cnidaria, Budding, Morphogenesis, Hydra
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Introduction |
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Here, we present data on a cnidarian FGFR-like RTK, Kringelchen, which has been isolated from the freshwater polyp Hydra and shares typical features with the FGFR of higher metazoa. Functional data indicate that Kringelchen is essential for boundary formation and tissue constriction, which are prerequisite for proper bud detachment.
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Materials and methods |
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Sequence analysis and database searches
The DNA sequence was determined by primer walking using an
35S-based sequencing kit (Amersham). Translation of the nucleotide
sequence into amino acids, mapping, determination of the pKa and homology
searches were performed with the HUSAR program package (German Cancer Research
Institute, Heidelberg). For phylogenetic analysis, a recently established
database was used, which is available under
http://pbil.univ-lyon1.fr/RTKdb
(Grassot et al., 2003).
Treatment with SU5402
Sixty animals per experiment were incubated in a final concentration of 10
µM SU5402 (Calbiochem), 1 mM ATP and 0.1% DMSO in Hydra medium for
24 hours. Control incubations were performed in the same solution without
SU5402. The stock solution of SU5402 (10 mM in DMSO) was stored in aliquots at
20°C. Fresh ATP solution was prepared for each experiment.
Antisense experiments
Mixed phosphorothioate antisense oligonucleotides were synthesized and
HPLC-purified by Eurogentec. They were designed for optimal function according
to Brysch and Schlingensiepen (Brysch and
Schlingensiepen, 1994): Phosphorothioate oligonucleotides
complementary to the 5' coding region of the kringelchen cDNA
were: oligo 1 (165-182 corresponding to nucleotides 65-82 of the coding
region, thioated nucleotides are marked with an asterisk) AAT
T*A*A* CTG GCT CT*G
T*A*A; and oligo 2 (217-234 corresponding to position
117-134 of the coding region) TGA A*G*C*
T*GG CAG TA*G A*T*C*.
In the semi-random mismatch controls nucleotides in 3 or 4 positions were
exchanged for each other. This prohibits specific interaction without changing
the base composition and is preferable to sense controls. In mismatch control
Oligo 1c, for example, nucleotide 3 was changed to position 6, 6 to 10, 10 to
14 and 14 to 3 (the exchanged nucleotides are underlined): AAC
T*A*T* CGT ACT
CG*G T*A*A*. In
mismatch control Oligo 2c, nucleotide position 4 was changed to 7, 7 to 12 and
12 to 4: TGA G*G*C*
A*GG CAA TA*G
A*T*C*. The rationale for the complex design
of the four antisense oligonucleotides was as follows. Three unmodified
nucleotides at the 5' end allowed phosphorylation to control the quality
of the oligonucleotides in a 20% sequencing gel. The next three or four
nucleotides (4-7) were phosphorothioated to protect against nuclease activity,
the next 6 or 7 nucleotides (7-13) were left unmodified to allow for RNase H
cleavage of the resulting hybrid, nucleotide 15 was left unmodified to avoid a
long stretch of phosphorothioates, which is known to increase unspecific
stickiness, and the last 3 nucleotides (16-18) at the 3' end were
thioated again to protect from nuclease digestion. The antisense
oligonucleotides were electroporated in 20 whole animals (100 µl volume, 4
mm cuvettes) using an Easyject Plus Electroporator (Equibio) at the following
settings: 1500 µF, 400 V, 99
. After electroporation, polyps were
stored at 4°C overnight and thereafter kept at 18°C. From 5x20
electroporated polyps (three independent experiments) the 39 survivors were
pooled for Fig. 4H. The
electroporation protocol was adapted from Lohmann et al.
(Lohmann et al., 1999
) and
yielded consistent results, while incubations with DOTAP (Boehringer) were
less reliable.
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Results |
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The features of the extracellular, ligand-binding domain of this ancient FGFR-like protein are particularly interesting: Usually, three Ig-like loops are present: the function of D1 is still unclear, D2 and D3 bind FGF, and D3 conveys FGF binding specificity. In Hydra, Ig-like loops D1 and D2 are likely to be formed by four highly conserved cysteines (positions 43, 94, 147 and 201; Fig. 1B,C). As in higher evolved FGFR, the intervening region is acidic (pKa=4.14), but the acidic residues are not clustered. Most conspicuous, however, is the region corresponding to D3: although usually cysteines clamp Ig-like loops, the alignment indicates that in the case of the putative Kringelchen D3 two hydrophobic amino acids might take this role, i.e. Phe324 and Tyr250.
Southern analysis (Fig. 2A) shows that kringelchen is encoded by a single copy gene. On a northern blot (not shown), a single mRNA about 3.8 kb in size was detected. The discrepancy between mRNA size and the obviously full-length cDNA (Fig. 1A) indicates that internal poly(T) priming might have occurred in the 3' UTR.
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Taken together, Kringelchen is a very good candidate for an archetype FGFR with highly conserved, but also clearly distinct, features as compared with higher evolved FGFR in triploblasts.
Kringelchen is dynamically expressed in all phases of bud evagination, differentiation and detachment
Well-fed Hydra propagate mainly asexually by budding. Bud
formation starts with the determination of a bud field in the mid gastric
region, followed by recruitment of tissue from the gastric column, which is
displaced into the evaginating early bud tissue. About 24 hours after the
first signs of bud evagination, head structures start to form in the apical
part. Shortly thereafter, the bud base begins to contract, foot tissue is
formed and, finally, about 4 days after evagination, a fully differentiated
young polyp detaches from the parent [for a detailed morphological analysis of
the budding process and staging see Otto and Campbell
(Otto and Campbell, 1977)]. As
the budding process activates the main developmental programs of a
Hydra, expression screening using whole-mount in situ hybridization
on budding polyps is an easy-to-use tool to isolate genes involved in
morphogenesis. Kringelchen was identified as a potentially
interesting gene by its astonishingly dynamic RNA expression pattern, which
distinguishes five phases (Fig.
3).
Early evagination phase
Until stage 3 (up to 3 hours after evagination) kringelchen RNA is
detectable only at the evagination site, from which the bud develops. At stage
2, kringelchen RNA is localized in a spot of about 60 epithelial
cells (see Fig. 3Q), then
spreads proximally and is, at stage 3, expressed in the ecto- and endoderm of
the growing protrusion (Fig.
3A,B). Expression of kringelchen is always restricted to
epithelial cells (see Fig.
3R,S).
Middle evagination phase
When the bud starts to elongate (stage 4), a spot-like zone of strong
expression forms in the tip endoderm. Only a low level of RNA is detected in
the growing body column (Fig.
3C-E). This changes as soon as the length of the bud approaches
its vertical extension: from now on, kringelchen RNA is additionally
found to be upregulated in a ring of ectodermal cells surrounding the bud base
(Fig. 3E-L,P).
Late evagination phase
In stage 5, when the bud length exceeds its vertical extension, the signal
in the ring-like zone intensifies and co-exists for a short time, together
with the apical patch of expressing cells
(Fig. 3G,H). By close-up
microscopy of three bud bases, the mean number of ectodermal cells per ring
was determined to be 171.4±33.2.
Differentiation phase
As soon as the earliest signs of tentacle buds are visible (stage 6, about
20 hours after evagination), the kringelchen signal is completely
lost from the apical tip (Fig.
3I,K). The circular zone of expression surrounding the now
narrowing bud base, remains intensive for the next 3 days
(Fig. 3I-L) and is about five
or six cells thick. Both parent and bud tissue express the gene in the
ectoderm until shortly before detachment
(Fig. 3K), when the ring has
narrowed to two or three cells (Fig.
3L). Sections (2 µm) showed that shortly before detachment
endoderm, mesogloea and ectoderm of the parent on the one side, as well as
endoderm and mesogloea of the bud on the other side form closed sheets. The
bud ectoderm, by contrast, is not yet closed at the prospective foot end and
columnar cells sit `waiting', well arranged and right above the detachment
zone (see arrowhead in Fig.
3L).
Detachment phase
When the bud is ready to detach (as seen by the narrow tissue bridge
connecting it to the parent), kringelchen RNA is no longer detectable
in bud tissue by in situ hybridization, but persists in the parent
(Fig. 3L). After detachment
(Fig. 3M), about 40
kringelchen-expressing cells remain as a small ring in the ectoderm
of the parent polyp for a short time. The ring-like expression zone constricts
quickly to a patch of cells (Fig.
3N-O) and within 1-2 hours expression is switched off
first in single cells, then in all of them.
As new buds always form above old ones, polyps with one new and one recently detached bud show the early diffuse expression in evaginating buds plus a ring or a patch of expression in a more basal position (Fig. 3I).
Detachment of the young polyp seems to leave a crater in the ectoderm of the parent (Fig. 3M): the ring of kringelchen-positive cells is elevated above the level of tissue within this ring. This raised the possibility that the detaching bud partially removes the ectoderm of the parent leaving the naked mesogloea behind. Detailed analysis of the detachment site in 2 µm sections showed, however, that the detachment site is covered by flattened ectodermal cells, which might in the following contract and close the `gap' to bring the kringelchen-positive cells back in the normal tissue environment.
The dynamic expression pattern could be caused by overlapping detection of two very closely related kinases. Control experiments with separate antisense RNA probes for the extra- and the intracellular parts of kringelchen yielded identical results, which excludes the possibility of crossreactivity with a related sequence.
In situ hybridization to single cell preparations (Fig. 3R,S) detects kringelchen expression exclusively in ecto- and/or endodermal epithelial cells (localization depending on the bud stage, see above). Overexposure detects kringelchen in up to 40% of the epithelial cells, which explains why RT-PCR detects transcription of the full-length sequence of kringelchen at a low level all over the body (not shown).
Correct kringelchen expression is tightly coupled to proper bud detachment
The dynamic expression pattern in morphogenetically active regions during
all phases of budding suggested that kringelchen participates in
general bud development. In order to analyse if interference with bud
detachment alters the expression pattern, we performed a competition
experiment: early budding and head regeneration compete for resources, which
is indicated by the fact that the growing bud prohibits head regeneration and
takes over the axis without detaching
(Tardent, 1972). Therefore, we
investigated the kringelchen RNA expression pattern in regenerating,
budding polyps. Either head or foot regeneration of the parent was induced by
dissection of the polyp right above or below the developing bud
(Fig. 4A,H). Polyps of our
Hydra vulgaris strain bearing buds up to stage 4 are unable to
regenerate a head if the head and upper body region of the parent are removed
immediately above an early (stage 3) bud
(Fig. 4A). The bud takes over
the axis of the parent in up to 100% of the cases. For a detailed analysis of
the kringelchen expression pattern in such secondary axes, budding
polyps were dissected early (stage 3) and late (stage 8).
Figure 4B shows that the early
RNA expression in the bud tip remains unaffected by regeneration of the parent
Hydra. But once the bud reaches stage 5-6, when usually the ring of
kringelchen-expressing cells forms at the bud base, this circular
expression zone was found to be severely distorted, broken up into patches or
even completely missing (Fig.
4C-G). In addition, many polyps express kringelchen RNA
at a high level throughout the bud tissue
(Fig. 4F), where normally no
expression is detectable. This feature seems to be due to the regeneration
process rather than being relevant for the lack of detachment, because it was
also found in buds dissected at stage 8, in which close to 90% of the polyps
detach and parent head regeneration occurs normal.
From our experiments, it appeared that proper kringelchen
expression and bud detachment are tightly linked. Because, in contrast to head
regeneration, foot regeneration does not inhibit bud detachment
(Tardent, 1972), we removed
the foot of budding polyps right below an emerging bud and performed in situ
hybridization as a control to validate the specificity of the observed
effects. The developing young polyps detached completely normally and even
slightly earlier (Fig. 4H). The
ring of kringelchen-expressing cells remained unaffected and
developed in its normal shape (Fig.
4I-L). Interestingly, the hybridization signal appeared more
intense and the ring was often thicker than in the normal budding polyps
which perhaps indicates a correlation with the faster detachment. In
addition, ubiquitous ectopic expression was never detected in buds developing
at foot regenerating parents, which indicates that only apical regeneration of
the parent induces `flooding' of bud tissue with kringelchen RNA.
This experimental control therefore confirms a tight relation between the
integrity of the ring-shaped kringelchen expression zone and normal
bud detachment.
Inhibition experiments reveal that Kringelchen functions in bud detachment
The dynamic expression pattern of kringelchen initially suggested
multiple functions during axis formation, patterning, differentiation and
detachment of a bud. In order to determine its function, we used two
approaches based on (1) the specific biochemical inhibition of FGFR kinase
activity, SU5402; and (2) inhibition of kringelchen translation by
phosphorothioate (PT) antisense oligonucleotides. Both treatments specifically
inhibited bud detachment (Fig.
5).
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SU5402 was also used in a treatment of polyps, which were `pregnant' with buds. Our feeding regime, in which polyps are fed only five times a week, partially synchronizes the culture: after 2 days of starvation, mainly stage 8-10 buds are present, which detach on the second day of feeding resulting in a culture containing mainly big, budless polyps, which are already induced to form new buds by feeding (`pregnant'). Budding is resumed on the third day of feeding by about 80% of the big polyps. Incubation of big, budless polyps (selected from the culture on the second day of feeding) for 24 hours in SU5402 had no effect on later bud development and detachment (n=100, control polyps; n=100, data not shown). None of the young polyps had developed beyond stage 5, when the SU5402 treatment was finished. Therefore, the decision to form a foot at the bud base takes time the inhibitor needs to be present between stage 3 and 7 to show maximal effects.
Like SU5402, phosphorothioate (PT) antisense oligonucleotides specific for the kringelchen mRNA prevented detachment if electroporation was started at stage 3 (Fig. 5G,H). Despite the low survival rate after electroporation, the effect was specific. The data given in Fig. 5G is derived from three experiments with 20 polyps each, the 39 surviving polyps were evaluated. The abnormality is similar to the one described for SU5402, with the exception that the young polyps are slim and elongated (up to twice as long as normally), indicating that proportioning is affected as well (Fig. 5H). In both cases, the circular expression domain of kringelchen is distorted or missing (Fig. 5D-F,H) comparable with the above-described bud-head competition experiment. Two scrambled PT-oligonucleotides (n=60 each) had no effect.
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Discussion |
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Kringelchen sequence features suggest that it is an ancient FGFR
Characteristic features for FGFRs of triploblasts are: (1) an intracellular
split tyrosine kinase domain and docking sites for downstream signalling
cascades, (2) a single transmembrane domain and (3) three Ig-like loops (Ig
I=D1 to Ig III=D3) in the extracellular, ligand-binding domain. Loops I and II
are separated by an acidic stretch of amino acids, which carries a consensus
sequence for CAM binding in vertebrates
(Burke et al., 1998). The third
Ig-like loop is optimized in vertebrates by alternative splicing for specific
binding of one of the 22 FGF ligands
(Ornitz and Itoh, 2001
).
The putative intracellular domain of Kringelchen is highly conserved with a
single transmembrane and a split kinase domain. Phylogenetic analysis of the
latter places Kringelchen at the base of the FGFR. Docking sites relevant for
downstream signalling are two SH2-binding domain consensus sequences, which
are prerequisite to couple to the SH2 domain of PLC
(Mohammadi et al., 1991
) and a
SH3-binding domain consensus for coupling to PI3-kinase. Thus, Kringelchen has
the potential to activate the PLC
/PKC cascade, which constitutes
(together with the Ras/MAPK und PI3-kinase pathways) the main FGFR downstream
signalling systems. Interesting in this respect is the observation that
Hydra PKC2 (HvPKC2) (Hassel et
al., 1998
) is co-expressed with kringelchen early in the
evaginating bud. No expression data are available for Hydra
PLC
yet (Koyanagi et al.,
1998
), but the preconditions for signalling through the Ras/MAPK
pathway are given, as two ras-related genes have been identified by
Bosch et al. (Bosch et al.,
1995
).
In the putative extracellular domain of Kringelchen, about 35% of the amino acids corresponding to Ig-like loops I, II and III of higher metazoan FGFR are identical to vertebrate and invertebrate receptors. The spacing and amino acid surrounding of four of the five extracellular cysteines in Kringelchen allow a clear assignment to the cysteines, which covalently clamp the D1 and D2 loops of higher metazoan FGFR (Fig. 1). As in other FGFRs, the region between D1 and D2 is acidic (pKa 4.41), but does not show a strong clustering of acidic amino acids.
The most conspicuous deviation from known FGFR is the lack of cysteines
between the putative Ig-like loop II and the transmembrane domain, which
excludes formation of a covalently linked D3. This feature is remarkable and
raises the question if Kringelchen is able to bind FGF. D3 is crucial in
vertebrates to confer specificity of FGF binding and together with D2
activates FGFR by FGF-induced dimerization. Point mutations in one or both
cysteines of D3 cause ligand-independent dimerization (and constitutive
activation), or generate FGFR with strongly reduced activity
(Burke et al., 1998). The
alignment in Fig. 1C reveals
that the positions corresponding to the D3-clamping cysteines are taken in
Kringelchen by Tyr249 and Phe324. Both amino acids are flanked by hydrophobic
residues, which provide the structural precondition for a potential
hydrophobic clamp. It is, thus, not excluded that even without cysteines a
third Ig-like loop forms in Hydra. Interesting under the evolutionary
aspect is that the Cys codons (TGT, TGC) can be
generated by a single point mutation in position 2 of the codons for either
Tyr (TAT, TAC) or Phe (TTT, TTC).
With the transition from an ancient AT-rich genome, as found in Hydra
(Galliot and Schummer, 1993
),
to the GC-rich genomes of higher organisms; a codon switch from Phe or Tyr to
Cys in this critical position might have improved the performance of FGFR. A
recently described platyhelminth protein that aligns with FGFR extracellular
and transmembrane domains also lacks D3
(Cebria et al., 2002
), but does
not show a comparable amino acid arrangement.
The question, `Do Hydra use FGF ligand(s) as signalling
molecules?' cannot be answered yet, but a comparison of conserved residues
identified in the crystal structure of vertebrate and invertebrate FGFR as
interaction sites with FGF (Plotnikov et
al., 1999; Nagendra et al.,
2001
) indicates a high level of conservation: of the 10 amino
acids identified as binding partners for FGF2 in the D2 and D3 loops of the
human FGFR1, Pro285, His286 and Asn345 are identical in Hydra (Pro
257, His258, Asn328); conserved exchanges are found in three more positions.
The remainder is neither conserved in the protostome invertebrates nor in
Hydra.
Consensus sequences that allow binding to other known extracellular FGFR
interaction partners, namely heparan sulfates, which enhance FGF binding
(Ornitz, 2000), or cell
adhesion molecules (CAM), are not conserved in Kringelchen. Of the eight
(mostly basic) amino acids identified in heparin binding, only one is present
(Nagendra et al., 2001
). An
HAV motif, which is a hallmark of CAMs and found in the vertebrate FGFR
adjacent to the acidic domain (Plotnikov
et al., 1999
), is missing in Hydra as well as in
Drosophila. Thus, as deduced from its sequence, Kringelchen might
bind FGF, but it is unlikely to function in a CAM-dependent manner, and it is
highly questionable if Kringelchen binds heparan sulfates.
Kringelchen indicates changing positional values
It is striking that kringelchen RNA is detectable in regions only
in which the positional value changes or from which adjacent tissue is
organized: the bud tip is kringelchen-positive only as long as it organizes
the axis (for a review, see Meinhardt,
1993); expression is switched off, when the positional value of a
mature head is reached and tentacle buds form. The bud base is positive, as
long as the positional value decreases to allow constriction and foot
formation, but mature foot tissue is negative. Finally, the ring, which
transiently persists in the parent after detachment, is positive until the
cells have taken their normal positions. This expression pattern indicates
that Kringelchen is involved in morphogenesis, but not in maintenance of
structures. It fits well a recently published mathematical model for pattern
formation in concentric rings (Berking,
2003
), which predicts expression patterns in the growing bud
similar to the kringelchen pattern.
Evidence for an evolutionarily conserved role of FGFR in boundary formation
The dynamic expression of kringelchen resembles FGFR expression in
the development of higher metazoa, where quick changes allow signalling
through particular pairs of FGF/FGFR in a locally restricted manner
(Ford-Periss et al., 2001).
Comparison of budding to such morphogenetic processes reveals parallels to (1)
branching morphogenesis (i.e. early budding) and (2) formation of boundaries
(i.e. late budding, detachment). Relevant examples for branching morphogenesis
are the formation of the tracheolar system in insects
(Skaer, 1997
) or of limbs in
vertebrates (Gorivodsky and Lonai, 2003). While formation of the tracheolae
requires the presence of FGF-secreting chemoattractant cells in the
surrounding tissue, Hydra bud formation resembles mechanistically
limb formation, where FGFR-based reciprocal inductive events control
evagination from an existing axis, tissue movement, elongation and later, by
coupling to downstream Wnt and PKC signalling, also differentiation and
establishment of the complex limb bud pattern (for a review, see
Wilkie et al., 2002
). It is
suggested that the Hydra Wnt homologue, HyWnt, and a PKC
isoform, HvPKC2, form a synexpression group with kringelchen
in the bud tip (Hobmayer et al.,
2000
; Hassel et al.,
1998
).
Despite these parallels, our inhibition experiments indicate that Kringelchen/FGFR signalling is, in contrast to the above-mentioned branching systems, neither involved in bud (i.e. branch) induction per se, nor the decisive factor for elongation and apical patterning. As Hyß-Cat and HyTcf as members of the Hydra Wnt pathway are detected already in the bud induction phase, when kringelchen is still silent, it remains to be shown if the interconnection of signalling cascades is different in Hydra or if redundancy of signalling cascades ensures proper bud formation.
Our experiments indicate that the evolutionary conserved function of FGFR
signalling, lies in boundary formation, which in the case of Hydra
budding is necessary to set the stage for constriction and foot formation at
the bud base. Boundary formation is an important FGFR function in vertebrates:
FGFR control early patterning along the anteroposterior and dorsoventral axis
of the brain, and are later essential to establish the midbrain-hindbrain
boundary (Altmann and Brivanlou,
2001). A recent report further corroborates this hypothesis,
because the transient knockout of Nou Darake in the platyhelminth
Dugesia led to a posterior shift of brain structures indicating a
defective boundary (Cebria et al.,
2002
). Nou Darake is a transmembrane protein, with high similarity
to the extracellular domain of FGFR, but lacks Ig-like loop III and a tyrosine
kinase domain.
The mechanism by which Kringelchen inhibition prevents detachment is very
likely to be complex. In SU5402-treated buds, the normal kringelchen
expression zone is distorted or missing, thus the inhibitor cannot directly
interfere with protein at the bud base and thereby inhibit detachment. RT-PCR
detects kringelchen expression all along the body column at a very
low level, and as FGFRs are often upregulated by autocatalytic loops and then
either act in a morphogen- or a threshold-like mode
(Hajihosseini et al., 2004),
our working hypothesis is that SU5402 inhibits Kringelchen upregulation by
blocking an autocatalytic loop close to the bud base. The crucial phase for
this inhibition is between bud stage 3 and 7.
These conclusions are, of course, valid only if SU5402 inhibits Kringelchen
as specifically as it inhibits other FGFRs, which has to be shown
experimentally in the future. In previous reports, non-detaching buds resulted
from treatment with more nonspecific protein (tyrosine) kinase inhibitors
(Perez and Berking, 1994;
Fabila et al., 2002
) or with
lithium ions (Hassel and Berking,
1990
). Strong arguments can now be made in favour of an
FGFR-specific effect. First, we obtained almost identical abnormalites using
SU5402 and kringelchen antisense inhibition; second, SU5402 is
described as highly specific for FGFR with no activity in insulin or EGFR, and
only weak reactivity with PDGFR in sixfold higher concentrations as used in
this study. Moreover, seven out of eight residues identified as important for
SU5402-FGFR binding (Mohammadi et al.,
1997
) are identical in Hydra. Non-FGFR tyrosine kinases
lack at least three (VEGFR) out of these eight crucial residues. And last but
not least, Southern, northern blots and the Hydra EST databases do
not provide evidence for another, closely related Hydra FGFR, which
might be the target for inhibition. We therefore conclude that the described
Kringelchen inhibition effects are specific and identify a role in boundary
formation as evolutionary conserved function of FGFR.
In the near future, it will be very interesting to investigate, which
ligand(s) activate Kringelchen with its peculiar structural properties.
Although it seemed for a long time as though peptides might function in
cnidaria like the complex growth factors in triploblasts (for a review, see
Bosch and Fujisawa, 2001), data
are accumulating from cnidarian EST projects that Hydra, like all
higher evolved metazoa, possesses growth factor homologues and their potential
receptors. It will be interesting to elucidate the relationship of peptide and
growth factor signalling in cnidarian morphogenesis.
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ACKNOWLEDGMENTS |
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