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First published online 1 February 2006
doi: 10.1242/dev.02265
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1 Molecular Evolution and Genomics, Heidelberg University, Im Neuenheimer Feld
230, 69120 Heidelberg, Germany.
2 Molecular Cell Biology, Darmstadt University of Technology, Germany.
3 Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg,
Germany.
4 Klinik für Tumorbiologie an der Universität Freiburg, Breisacher
Strasse 117, 79106 Freiburg, Germany.
5 Center for Integrative Bioinformatics Vienna (CIBIV), Max F. Perutz
Laboratories (MFPL), Dr Bohr Gasse 9, 1030 Vienna, Austria.
6 University of Veterinary Medicine, Vienna, Austria.
7 Medical University, Vienna, Austria.
8 Vienna University, Vienna, Austria.
9 Institut für Zoologie und Limnologie, Universität Innsbruck,
Technikerstrasse 25, 6020 Innsbruck, Austria.
* Author for correspondence (e-mail: holstein{at}zoo.uni-heidelberg.de)
Accepted 28 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Dickkopf, dkk, Wnt signalling, Wnt antagonism, Regeneration, Axis formation, Neurogenesis, Cnidaria, Hydra, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Nelson and Nusse,
2004; Nusse,
2003), and their appearance in early metazoan evolution was
probably linked to the origin and evolution of multicellular animals
(Kusserow et al., 2005). In
vertebrate embryonic development, Wnt ligands initiate signalling by
interacting with two types of receptor molecules: the seven-pass
trans-membrane protein Frizzled and the low-density lipoprotein receptor
Lrp5/Lrp6. It is currently assumed that Wnt ligands bind to both Lrp5/Lrp6 and
Frizzled to form a functional ligand dual-receptor complex that activates the
canonical Wnt-ß-Catenin pathway. Dickkopf 1 (Dkk1), a major secreted Wnt
antagonist, binds to Lrp5/Lrp6 and its coreceptor Kremen to inhibit Wnt
signalling (Davidson et al.,
2002; Mao et al.,
2002; Mao et al.,
2001; Semenov et al.,
2001). Inhibition of canonical Wnt signalling by Dkk1 is essential
for head formation and limb patterning in the vertebrate embryo
(Davidson et al., 2002;
Glinka et al., 1998;
Grotewold and Ruther, 2002b;
Hashimoto et al., 2000;
Kazanskaya et al., 2000;
Mukhopadhyay et al., 2001;
Shinya et al., 2000).
Of the four Dickkopf (Dkk) protein family members, Dkk1, Dkk2 and Dkk4 are
able to inhibit Wnt signalling (Glinka et
al., 1998; Krupnik et al.,
1999; Mao and Niehrs,
2003; Wu et al.,
2000). In addition, Dkk2 can act as an Lrp6 agonist, as well as
antagonist, depending on the cellular context
(Mao and Niehrs, 2003;
Wu et al., 2000). Dkk3 is
unique within the Dickkopf family in that it is not able to antagonize Wnt
signalling (Krupnik et al.,
1999; Mao and Niehrs,
2003).
Vertebrate Dickkopf molecules consist of two cysteine-rich domains (CRD1
and CRD2), which are separated by a spacer region, diagnostic for grouping of
Dkk proteins. It has been shown that CRD2 is necessary and sufficient to
repress canonical Wnt signalling by competing with the Wnt-Frizzled complex
for binding to the Lrp5/Lrp6 receptor. CRD1 is thought to have a modulating
function on CRD2 (Brott and Sokol,
2002; Li et al.,
2002).
Hitherto, it is completely unknown at what point in metazoan evolution
Wnt-Dickkopf antagonism was established. Although the receptor proteins
Frizzled and Lrp5/Lrp6 from vertebrates have homologues in insects, no
Dickkopf protein could be identified in the insect and nematode genomes. A
Dkk3-related protein was recently identified in Hydra
(Fedders et al., 2004), and it
was suggested that Dkk3 represents the ancestral Dickkopf type. It was
furthermore proposed that subsequent genome duplication created the vertebrate
Dkk1/2/4 subfamily (Fedders et al.,
2004). According to this proposal, the Dickkopf-Wnt antagonism was
established relatively late in metazoan evolution, i.e. concomitant with the
radiation of vertebrates.
We describe here a new Dickkopf-related molecule from the freshwater polyp Hydra and the starlet sea anemone Nematostella. The novel Hydra Dickkopf protein was isolated as regeneration specific in a yeast signal peptide secretion screen. Structural and phylogenetic analysis indicates that this dkk gene is a dkk1/2/4 homologue, from which the vertebrate Dkk1, Dkk2 and Dkk4 arose by gene duplication. Moreover, our expression and functional analyses support the view that the Wnt-Dickkopf antagonism is phylogenetically very old, and probably had a major function in setting up a neurogenic environment in the body column of the pre-bilaterian Hydra.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
as well as Hydra vulgaris Basel and AEP
(Martin et al., 1997), were
cultured at 18°C (Takano and Sugiyama,
1983), fed five times a week, and used 24 hours after feeding for
experiments. Gametogenesis was induced in polyps of Hydra vulgaris
strain AEP as described (Rentzsch et al.,
2005).
Elimination of interstitial cell lineage
Temperature-sensitive interstitial cells of Hydra magnipapillata
strain sf-1 were eliminated by culturing polyps at 28-30°C for 5 days;
afterwards starved polyps were kept at 18°C for up to six weeks (see
Results). Loss of interstitial cells and derivatives was examined in macerates
(David, 1973).
Regeneration and wounding experiments
Polyps were bisected at 20%, 50% or 80% body length by using a small
scalpel. In some experiments, heads were removed without injury by tying a
knot with a thin hair around the subhypostomal region of the polyp as
described (Newman, 1974). This
caused the head to be gradually pinched off without leaving an open wound with
exposed endoderm. For wounding, one deep cut was set into the middle of the
body column (30% body width). All animals were transferred into fresh medium
after treatment.
Alsterpaullone treatment
Alsterpaullone (Calbiochem) was dissolved in DMSO and diluted with hydra
medium to 5 µM. Daily fed polyps were incubated in alsterpaullone for 24 or
48 hours.
Molecular techniques
Isolation of mRNA
We isolated polyA+ RNA from total RNA
(Suzuki et al., 2001) of
Hydra vulgaris strain Basel, Hydra magnipapillata strain
sf-1 (nf-1), and Hydra magnipapillata strain 105, by using the
PolyATtract mRNA Isolation System III (Promega).
Cloning of hydkk1/2/4
A hydkk1/2/4 cDNA clone was isolated in a screen for
organizer-specific transcripts from the i-cell depleted Hydra
magnipapillata strain sf-1 by using the signal peptide secretion approach
(Fig. 1)
(Jacobs et al., 1997;
Jacobs et al., 1999). Blast
search revealed a match with clone taa05h01 (CA303262) from the hydra EST
collection
(http://mpc.uci.edu/hampson/public_html/blastlif9).
This sequence contains a complete polyA tail. The full-length sequence was
amplified from Hydra magnipapillata strain 105 cDNA by
5'RACE-PCR using the GeneRacer Kit (Invitrogen), and specific primers
(5'-CCGCAGAGTGCACCTTCTTTAACATAGCTATTACATTGC-3' and
5'-GCAGTCTGCATCCTTTTTGCAAGACTCGGC-3'). Complete
hydkk1/2/4 cDNAs were additionally amplified from both, Hydra
vulgaris strain Basel and Hydra magnipapillata strain sf-1 by
using oligo-dT primed cDNA and specific primers
(5'-GAAAACATACATCTTTTCTGATTTATCAATC-3' and
5'-(T)17ATAATTTAACTCG-3'). PCR amplicons were cloned into the
pGEM-T vector (Promega). A sequence comparison of the three strains revealed
that hydkk1/2/4 from Hydra magnipapillata sf-1 exhibited 10
nucleotide substitutions within the ORF, resulting in seven amino acid
exchanges. For all experiments the Hydra vulgaris clone was used.
Cell culture experiments
TOPFLASH assay in HEK293T cells was performed as previously described
(Wu et al., 2000).
Transformation of cells was carried out in 96-well plates, in triplicate, with
1 ng each of GFP, renilla and hfrizzled1, 10 ng TOPFLASH
vector, 8 ng mwnt1, 3 ng human LRP6, 20 ng
hydkk1/2/4, 5 ng xdkk1 and 3.125 ng hkrm1. Samples
were supplemented with pCS2+ vector to 100 ng DNA. After 24 hours,
the transformation efficiency was checked by monitoring GFP fluorescence.
Firefly luciferase activity was normalized against renilla.
In situ hybridization (ISH)
Whole-mount ISH was performed as described
(Grens et al., 1996;
Martinez et al., 1997) at a
probe concentration of 0.05 ng/µl for 36 hours. Double ISH was performed as
described (Hansen et al.,
2000; Philipp et al., 2004), with NBT/BCIP substrate followed by
Fast Red substrate. Sense and antisense riboprobes were produced from a 534 bp
fragment of hywnt3a (bp 624-1158) and from full-length
hydkk1/2/4 using the DIG or FITC RNA Labelling Kit (Roche).
Macerate ISH was performed by preparing a fixed cell suspension of
macerated cells (David, 1973)
dropped (100 µl) onto a poly-L-Lysine-covered object slide and allowed to
dry for 45 minutes at room temperature. After washing for 5 minutes with
PBS/PBSTween, preparations were pre-hybridized (2 hours, 60°C), hybridized
(24 hours, 60°C, 0.1 ng/µl probe) and washed (2xSSC, 2x5
minutes, 60°C; 2xSSC+0.1 % CHAPS at 50°C and 40°C), then
sequentially incubated with blocking reagent (3 hours at room temperature) and
anti-digoxigenin monoclonal antibody (4000-fold diluted, overnight 4°C).
Detection was carried out as described for whole-mount ISH.
In vitro transcription of hydkk1/2/4 and hywnt3a mRNA
The complete hydkk1/2/4 ORF was amplified and cloned as a
BamHI/ClaI fragment using the oligonucleotides
5'-CTTTTCGGATCCATCAAT-3' and
5'-GTATTTAAATCGATACAAAGATCCAC-3' into pCS2+ and pCS2+MT (myc-tag)
expression vectors (Rupp et al.,
1994). The hywnt3a ORF was amplified from Hydra
vulgaris cDNA as a ClaI/XbaI fragment using the
oligonucleotides 5'-CATCGATTTGCCGCCACCATGGGCACG-3', including the
KOZAK sequence, and 5'-TTTCTAGACTATTTACAGGTGTATTCAG-3', and cloned
into pCS2+ vector. PCR conditions for all amplicons were: 2 minutes at
94°C, then 30 cycles of 30 seconds at 94°C, 30 seconds at annealing
temperature and 1 minute at 72°C; the annealing temperature was 55°C
for the complete hydkk1/2/4 sequence, 46°C for the
hydkk1/2/4 ORF and 52°C for the hywnt3a ORF. Sequencing
was performed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) or by
the GATC Company (Konstanz).
Phylogenetic analyses
Protein sequence alignment was performed using the TCoffee alignment tool
(http://igs-server.cnrs-mrs.fr/Tcoffee)
and adjusted manually. Maximum likelihood trees were computed using IQPNNI
(Vinh and von Haeseler, 2004)
(300 repetitions) and quartet puzzling as implemented in TREE-PUZZLE 5.2
(Schmidt et al., 2001) (10,000 intermediate trees), both using the VAG model
of evolution.
The following sequences were used: HyDkk1/2/4-A; HyDkk1/2/4-B (EST CN559480); Nematostella vectensis Dkk1/2/4, Dkk3 (contigs 7341 and 9370); Acropora millepora (EST GS01bF09.b1); Branchiostoma belcheri tsingtaunese (EST AY608670); Zebrafish (Danio rerio) Dkk1 (BAA82135); Xenopus Dkk1, Dkk2 (AAC02427, XLA300197); mouse Dkk1, Dkk2, Dkk3, Dkk4 (NM_015789, NM_020265, NM_015814, NM_145592); human Dkk1, Dkk2, Dkk3, Dkk4 (BAA34651, BAA85465, BAA85488, BAA33475); Dictyostelium discioideum WGS_BC5V2_0 (Sanger Institute); human and mouse Colipase (Col) (AAP35458, AAL40731).
Microscopy
Specimens were analyzed using a Zeiss Stemi SV 11 binocular, a Zeiss
Axiovert 100, or a Nikon Eclipse-80i microcope, both equipped with
interference contrast. For microphotography, either a Diagnostic Instruments
Spot-II, a Canon PowerShot G5 or a Nikon DSL-1 camera were used. Micrographs
were processed using the manufacturer's software in combination with MetaMorph
or Adobe Photoshop software.
Xenopus experiments
In vitro fertilization, embryo culture, staging, microinjection and culture
of explants were carried out as described
(Gawantka et al., 1995). mRNA
was produced with the MessageMachine SP6 Kit (Ambion) from the
hydkk1/2/4 ORF and the hywnt3a ORF in the NotI
linearized pCS2+ vector; mRNA was purified with P6 Spin Columns (Bio-Rad).
Injections were done radially into all blastomeres of four-cell-stage
Xenopus laevis embryos (100 pg), or in case of hywnt3a
radially into ventral blastomeres (1 ng). Control mRNA (10 pg xdkk1,
12.5 pg xwnt8) was injected as well.
Animal cap assay
Xenopus laevis embryos were injected in the animal pole of all
blastomeres at the four-cell stage: xwnt8 (100 pg), xdkk1
(200 pg), hydkk1/2/4 (6 ng). Animal caps were explanted at stage 8-9
and analyzed by RT-PCR at stage 10 for the induction of siamois
expression. xbra expression validates mesoderm-free caps,
histone-4 expression was monitored for normalization.
Luciferase assays
Xenopus laevis embryos were injected in all blastomeres at the
four-cell stage, and subequatorially with the Wnt-reporter construct
siamois-luciferase p01234 alone or in conjunction with xwnt8 (150
pg), xdkk1 (300 pg) and hydkk1/2/4 (750 pg and 3 ng).
Embryos were collected at stage 10-10.5 in Passive Lysis Buffer (Promega) (25
µl/embryo). Results are presented as Relative Luciferase Units (RLU).
Secondary axes assay
Xenopus laevis embryos were injected into two opposite blastomeres
at the four-cell stage with wnt8 (12.5 pg), xdkk1 (10 pg)
and hydkk1/2/4 (1 ng).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Klein et al., 1996). Because
Marcum and Campbell (Marcum and Campbell,
1978) have shown that Hydra lacking nerve cells,
nematocytes and interstitial cells develop normally, we tried to eliminate the
highly abundant transcripts of this cell line. We used heat-shocked animals of
the mutant strain sf-1 from Hydra magnipapillata, which had lost
their interstitial stem cells and nematocytes. After inducing head
regeneration, we collected the heads and isolated regenerating stumps at
various time-points after head removal (0.5 to 24 hours), and prepared a
regeneration-specific cDNA library (Fig.
1A). The most abundant clone (10 %) in the signal peptide
secretion screen contained a 328 bp fragment encoding a cysteine-rich protein.
Using 5'RACE we completed the sequence obtaining a 395 bp transcript
with short 5' and 3'UTRs (88 bp and 31 bp, respectively). The open
reading frame encodes a protein of 73 amino acids, containing a cysteine-rich
motif and a signal peptide of 19 amino acids (see Fig. S1A in the
supplementary material). BLAST searches at NBCI revealed with highest score
(E-value 0.005) similarity to the CRD2 of vertebrate Dickkopf subfamily
members Dkk1, Dkk2 and Dkk4. The CRD2 motif of the Dkk family shares a high
structural similarity with colipases (coenzymes of lipases) and has been
assigned to the colipase fold (Aravind and
Koonin, 1998). However, the similarity of the novel Hydra
Dkk protein to colipases was much lower (E-value 0.62).
|
). Because the novel Hydra Dkk-related molecule
identified in the screen shows a higher similarity to CRD2 of the vertebrate
Dkk1/2/4 subfamilies, we hypothesized that it might be a new member of the Dkk
family in cnidarians. For a better overview, we performed a search for members
of the Dkk family using EST data from several other invertebrates. We found
ESTs from two other cnidarians, Nematostella vectensis and
Acropora millepora, from the urochordate Branchiostoma belcheri
tsingtaunese, and from the cellular slime mold Dictyostelium. No
Dkk-like proteins were found in insect and nematode genomes. The Nematostella contigs encode for two different Dkk-like proteins, each comprising two complete cysteine-rich domains (see Fig. S1A,B in the supplementary material). Alignments using TCoffee (Fig. 2A), but also ClustalW and Muscle (data not shown), reveal a higher similarity of cnidarian Dkks to vertebrate Dkks than to vertebrate colipases. In cnidarian Dkks, all ten cysteine residues are completely conserved, and some other amino acids, like glycines and lysines, are partially conserved among the species. Vertebrate Dkk1, Dkk2, and Dkk4 share a number of motifs that cannot be found in the vertebrate Dkk3 subfamily. Cnidarian Dkks share a number of amino acids with both the Dkk1/2/4 and the Dkk3 subfamilies, but again not with the colipases (Fig. 2A).
The IGPNNI ML-tree (Fig. 2C) of the CRD2 shows two distinct cnidarian Dkk subtrees: one containing the more dkk3-like sequences, the other containing the Hydra Dkk-related protein isolated in the secretion screen and the second nvdkk. The latter subtree is positioned closer to the highly supported cluster of the vertebrate Dkk subfamilies 1, 2 and 4. Unfortunately, neither TREE-PUZZLE nor bootstrap analyses were able to resolve any of the inner branches due to the high divergence of the Dkk family. However, a basal position between the Dkk3 and the Dkk 1/2/4 subfamilies was never contradicted. For clarity, we refer to the novel cnidarian Dkks as NvDkk1/2/4 and HyDkk1/2/4 (see Discussion).
hydkk1/2/4 is expressed in endodermal derivatives of the interstitial stem cell lineage
In order to unravel the function of the putative hydkk1/2/4 gene,
we analyzed its expression pattern by in situ hybridization. Strikingly,
Hydra's head was completely free of hydkk1/2/4-expressing
cells, whereas the entire body exhibited a strong endodermal expression with a
sharp boundary below the tentacle formation zone
(Fig. 3A,B) (see also
Hobmayer et al., 1990a;
Hobmayer et al., 1990b). In
many specimens, we found a graded expression, diminishing towards the peduncle
region. This expression pattern is inversely related to that of hywnt3a,
hytcf, brachyury and other head-specific genes
(Hobmayer et al., 2000;
Technau and Bode, 1999).
hydkk1/2/4 is not expressed in endodermal epithelial cells, but is
present in gland cells. Gland cells are derivatives of the interstitial stem
cell lineage (i-cells), which also gives rise to nerve cells, nematocytes and
sex cells. To verify the i-cell nature of hydkk1/2/4-expressing gland
cells, we investigated the hydkk1/2/4-expression pattern in the
temperature-sensitive Hydra magnipapillata strain sf-1. This mutant
loses interstitial stem cells after heat shock at 28°C and, over time, all
non-dividing derivatives, i.e. nematocytes and neurons, but also gland cells
disappear (Sugiyama and Fujisawa,
1978). As expected, hydkk1/2/4-expressing gland cells
were lost after a heat shock (Fig.
3C). Because gland cells have a limited proliferating capacity
(Schmidt and David, 1986),
they are lost more slowly than nematocytes and nerve cells. Thirty days after
heat shock, the number of hydkk1/2/4-expressing gland cells
diminished by more than 90% to about 50. These residual hydkk1/2/4
cells were full of vesicles (Fig.
3D). We also found a downregulation of hydkk1/2/4
expression in gland cells at sites of oogenesis
(Fig. 3E-G), but not
spermatogenesis (Fig. 3H). This
is in accordance with their i-cell origin, as oogenesis is accompanied by the
differentiation of i-cells into nurse cells.
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hydkk1/2/4 expression during regeneration
During regeneration the transcriptional regulation of hydkk1/2/4
is highly dynamic. In animals bisected at 80% body length, hydkk1/2/4
expression was markedly upregulated at the site of cutting within 30 minutes
(Fig. 4). High levels of
expression were sustained up to 6 hours after head removal in 50% of all
animals (Fig. 4B,C), which is
significantly longer than the time required for wound closure (1 hour).
Afterwards, hydkk1/2/4 was completely downregulated in the
presumptive head (Fig. 4B).
With the emergence of tentacles, 30 hours after head removal, the apical
region of all regenerates was free of hydkk1/2/4 expression.
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hydkk1/2/4 is involved in the injury response
The rapid increase of hydkk1/2/4 message during head regeneration
could be either caused by the removal of inhibiting signals emanating from the
head or by the injury stimulus itself. We tested both possibilities. (1)
Animals were injured by making a deep cut into the body column and leaving the
head intact. Injured animals exhibited a strong stimulation of
hydkk1/2/4 transcripts for at least 6 hours at the site of injury
(Fig. 5A). Even after wound
closure, increased levels of hydkk1/2/4 transcripts were still
recognizable in cells at the site of the wound. (2) Heads of polyps were
removed without wounding by tying a knot with a thin hair around the polyp's
subhypostomal region (Newman,
1974). No significant upregulation of hydkk1/2/4
expression occurred in such polyps (Fig.
5B). In agreement with Newman's observations, tied regenerates did
not regenerate normally. In conclusion, our results suggest that it is the
injury signal itself that causes a stimulation of hydkk1/2/4
expression, and not the removal of an inhibitory signal from the head.
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Figure 6 shows representative examples of 35-day-starved animals at 4 hours after head removal (Fig. 6B-D) and at 9 days of regeneration (Fig. 6E-G). Most regenerating pieces had no hydkk1/2/4+ cells (Fig. 6B), although a few had up to 50 hydkk1/2/4+ cells (Fig. 6C,D). Following regeneration, almost all `regenerates' lacked hydkk1/2/4+ cells and failed to regenerate properly. Very few pieces regenerated a head (Fig. 6G). Although most of these regenerates lacked hydkk1/2/4+ cells (Fig. 6G), we presume that such successful regenerates had hydkk1/2/4+ cells at the start of regeneration.
We also tested wnt expression in hydkk1/2/4-depleted animals. We found a significant number of ectopic hywnt3a expression domains in such animals (Fig. 6H-J), but never in non-heat-shocked control animals that were starved for the same time (data not shown). In 47% of all analyzed hydkk1/2/4-depleted sf-1 polyps (n=55), several different-sized patches of hywnt3a-expressing cells were formed along the body column. This suggests that in a hydkk1/2/4-depleted background hywnt3a expression can be spontaneously activated. These are unexpected data, indicating that hydkk1/2/4 gland cells are required for normal regeneration in Hydra and have a function in the regulation of hywnt3a gene expression (see Discussion).
Inhibition of hydkk1/2/4 expression in alsterpaullone-treated polyps
We tested the putative antagonism between Wnt and hydkk1/2/4 gene
expression in an additional experiment. We created a hywnt3a
overexpression situation by treating polyps with the kinase inhibitor
alsterpaullone (Knockaert et al.,
2002; Leost et al.,
2000). Alsterpaullone specifically inhibits Gsk3 in Hydra
and thereby activates the canonical Wnt signalling pathway
(Broun et al., 2005).
Alsterpaullone-treated polyps (24 hours) formed numerous spot-like
hywnt3a-expression domains after 3 days (see Fig. S2 in the
supplementary material), followed by ectopic tentacles after 3-4 days
(Fig. 7A,B) and ectopic
head-like structures after 8 days (Fig.
7A,B). hydkk1/2/4 transcription levels successively
decreased 24-48 hours after the onset of alsterpaullone treatment, and were
completely absent when ectopic tentacles were formed
(Fig. 7B,C). Double ISH
indicates that the downregulation of hydkk1/2/4 starts from the
centre of the hywnt3a-expression domains in the body column (Fig. S2
in the supplementary material). At later stages, hydkk1/2/4
expression was restored in the tentacle-free tissue between distinct heads
(Fig. 7C). Thus,
hydkk1/2/4 expression is negatively regulated by canonical Wnt
signalling.
|
|
), endogenous xdkk1 overexpression in the embryo
leads to enlarged head structures and reduced posterior structures (Dickkopf
phenotype) due to enhanced inhibition of late canonical Wnt signalling.
Injection of hydkk1/2/4 mRNA in each blastomere of four-cell embryos
resulted in severe morphological defects comparable to the Dickkopf phenotype
(Fig. 8A): anterior structures,
like the cement gland, were enlarged, whereas posterior trunk regions were
severely reduced if not abolished. To further demonstrate the Wnt-inhibitory
activity of hydkk1/2/4, we co-injected hydkk1/2/4 with
xwnt8, which induces secondary axis formation in Xenopus
embryos. This secondary axis formation could be blocked by the co-injection of
hydkk1/2/4, even though it was less effective than xdkk1
(Fig. 8B). These experiments
clearly show that HyDkk1/2/4 is a functional homologue of vertebrate Dkk1 and
Dkk4. To determine the inhibiting effect of HyDkk1/2/4 on downstream targets
of canonical Wnt signalling, an animal cap assay was performed using
siamois, a direct Wnt target gene, as readout
(Fig. 8C). Embryos were
injected with xwnt8 mRNA in presence or absence of xdkk1 and
hydkk1/2/4 mRNA. RT-PCR was then carried out on animal caps to
measure the expression of siamois. Detection of brachyury
and histone-4 served as controls for mesodermal contamination and
internal normalization, respectively. Non-injected control embryos showed no
detectable siamois transcripts. siamois was upregulated upon
xwnt8 injection, and this upregulation was abolished by co-injection
with xdkk1 or hydkk1/2/4. This result was further verified
using the TOPFLASH Wnt reporter (Korinek
et al., 1997) (Fig.
8D). In this assay, HyDkk1/2/4 showed a dose-dependent,
Wnt-inhibitory activity. These findings strongly indicate that HyDkk1/2/4 is a
Dickkopf homologue capable of inhibiting Wnt signalling.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Brott and Sokol,
2002). Here, we describe, for the first time in an invertebrate,
the identification of a novel Dickkopf-related protein, which has an
inhibitory function in Wnt signalling.
|
).
Based on their conserved CRD2, all cnidarian dkk genes share
features of chordate dkk3 and dkk1/2/4 genes. The
significant diversification of cnidarian dkk genes suggests a deep
split into dkk3 and dkk/1/2/4 gene families during early
metazoan evolution. Although this could not be clarified with certainty by
phylogenetic analysis because of the high divergence of the sequences, this
notion is supported by the chromosomal location of vertebrate Dkks. An
analysis of paralogy regions in the human genome using the paralogon database
Human 5.28 of Wolfe and McLysaght (Luke et
al., 2003; Lundin,
1993; McLysaght et al.,
2002)
(http://wolfe.gen.tcd.ie/dup/human5.28/)
shows that human DKK1, DKK2 and DKK4 are located within
related chromosomal regions (DKK1 maps to 10q11, DKK2 to
4q25 and DKK4 to 8p11). These sites lie within the well-characterized
4/5/8/10 paralogy group, which also contains FGF receptors and NK homeobox
genes (Birnbaum et al., 2000;
Coulier et al., 2000a;
Coulier et al., 2000b;
Leveugle et al., 2004;
Pollard and Holland, 2000),
and numerous other duplicated gene families. Genes within this paralogy region
were duplicated early in vertebrate evolution
(Pollard and Holland, 2000;
Luke et al., 2003). Hence, we
conclude that dkk1, dkk2 and dkk4 most likely originated by
gene duplication. By contrast, human DKK3 maps to 11p15.3, which is
not part of the same set of paralogy regions. This finding is consistent with
the hypothesis that the features common to the Hydra, Nematostella
and vertebrate dkk1/2/4 subgroup were likely to have been present in
the common ancestor of cnidarians and bilaterians.
hydkk1/2/4 is an early regeneration-responsive gene
The most obvious role HyDkk1/2/4 plays in Hydra is its function
during early regeneration. We found a rapid and dramatic increase of
hydkk1/2/4 message at the site of injury within the first hour after
head removal. hydkk1/2/4 upregulation was clearly related to the
injury stimulus, as it also occurred by simply cutting the animals at any site
in the body column. This early upregulation seems to be indispensable for
regeneration, because animals whose heads were removed by means of the
ligature technique (Newman,
1974), with minimal or even without injury, exhibit no
hydkk1/2/4 upregulation and could not regenerate normally.
Furthermore, animals that have lost hydkk1/2/4-expressing cells also
lose their regeneration capacity.
The regeneration deficient mutant strain reg-16 is also sensitive to the
injury stimulus: regenerates develop normally after setting a second cut at
the site of injury (Sugiyama and Fujisawa,
1977). We therefore presume that an essential trigger for head
regeneration in Hydra is the early release of Dickkopf proteins at
the site of cutting. This role of gland cells in patterning processes has
certainly been underestimated so far.
Interestingly, Prockop et al. (Prockop
et al., 2003) found in the process of mammalian tissue repair that
marrow stromal cells (MSCs) from the bone marrow secrete and require Dkk1 for
cell expansion in vitro and during the process of tissue repair. It probably
interacts with Wnt5a in the growth regulation of MSCs
(Gregory et al., 2005;
Gregory et al., 2003;
Horwitz, 2004;
Prockop et al., 2003).
Dkk1 has also been found to be strongly upregulated at the sites of
apoptosis during vertebrate limb development, and in UV-irradiated tissue
(Grotewold and Ruther, 2002a;
Grotewold and Ruther, 2002b).
Because regeneration in Hydra is accompanied by dramatic changes in
the pattern of cell cycle and proliferation at the site of cutting
(Holstein et al., 1991;
Holstein et al., 2003), we
propose that HyDkk1/2/4 in Hydra has a similar function in the
response to stress signals and the initiation of tissue repair. It is as yet
unclear which signal actually causes the extremely fast upregulation of
hydkk1/2/4 transcripts. One molecular trigger could be Jun (also
known as c-Jun), a stress-responsive transcription factor and activator of
dkk1 that is upregulated during embryonic wounding
(Grotewold and Ruther, 2002a;
Grotewold and Ruther, 2002b).
Another candidate is ß-Catenin, which can also activate dkk1
expression in a dose-dependent manner
(Gonzalez-Sancho et al., 2005;
Niida et al., 2004). Both
genes have been identified in Hydra
(Hobmayer et al., 2000).
Our data show that there exists an additional level of complexity in the
regulation of hydkk1/2/4 expression. We found during late
regeneration, i.e. in late bud stages and after activation of the
Wnt/ß-catenin pathway by treatment with alsterpaullone, a complete
silencing of hydkk1/2/4 expression. This clearly indicates that the
early activation of hydkk1/2/4 expression during regeneration is only
transient. The shift in the transcriptional regulation of hydkk1/2/4
found during Hydra regeneration shares similarities with the
transcriptional downregulation of dkk1 in human colon tumours. Colon
tumours exhibit activated Wnt/ß-Catenin signalling and downregulation of
dkk1 expression (Gonzalez-Sancho
et al., 2005). It was proposed that hypermethylation of the
dkk1 promotor leads to dkk1 silencing, similar to the
silencing of other Wnt inhibitory genes and to dkk3
(Suzuki et al., 2004;
Caldwell et al., 2004;
Gonzalez-Sancho et al., 2005).
Thus, in Hydra, hydkk1/2/4 expression appears to be stably silenced
by Wnt/ß-Catenin signalling in the head, while it might suppress
Wnt/ß-Catenin signalling in the body column, generating the distinct
compartments of Hydra's body, as implied by the sharp boundary
underneath the head.
HyDkk1/2/4 is an evolutionary conserved antagonist of Wnt signalling
We tested the function of HyDkk1/2/4 as a putative Wnt inhibitor in
Xenopus embryos. In this heterologous system, HyDkk1/2/4 has similar
Wnt-inhibitory properties to endogenous XDkk1. (1) HyDkk1/2/4 and the
endogenous XDkk1 have the same anteriorizing capacity in Xenopus
embryogenesis. (2) HyDkk1/2/4 can block XWnt8-induced secondary axis formation
(Fig. 8B). (3) HyDkk1/2/4
blocks the induction of the downstream target gene of the canonical Wnt
pathway siamois in animal cap assays. The fact that HyDkk1/2/4,
corresponding to the carboxy-terminal cysteine-rich domain of Dkks, can exert
an inhibitory effect on Wnt signalling is consistent with similar findings for
the CRD2 of vertebrates. It was also shown that the CRD2 of Dkk4 is
proteolytically cleaved from the full-length protein
(Krupnik et al., 1999), which
might represent an ancient feature of the basal Dkk4 within vertebrates
(Fig. 2C). By comparison, Dkk3
has a linker sequence between CRD1 and CRD2 without a proteolytic cleavage
site, and has not been shown to inhibit or modulate Wnt signalling at all
(Krupnik et al., 1999;
Mao and Niehrs, 2003).
The evolutionary origin of Dickkopf proteins remains unclear, but
cnidarians suggest a possible scenario. The CRD2 is similar to colipases,
which facilitate the interaction of pancreatic lipases with lipid micelles
(Krupnik et al., 1999). Based
on this structural similarity, it was proposed that the CRD2 of Dkk could help
other proteins to interact with lipids in order to regulate Wnt function
(Aravind and Koonin, 1998). Wnt
proteins are indeed tightly associated with the cell surface
(Nusse, 2003;
Smolich et al., 1993). Wnts
are palmitoylated proteins and are therefore much more hydrophobic than is
predicted from their primary amino acid sequences
(Nusse, 2003;
Willert et al., 2003). Thus,
lipid binding of Dkk may have initially served to tether Wnt ligands to target
membranes. From such a facilitated interaction of Wnt and Dkk at the plasma
membrane, the Dkk/Wnt antagonism may have evolved. In accordance with this
idea, we found that HyDkk1/2/4 enhances human LRP6- and mouse Wnt1-induced Wnt
signalling in 293T cells 1.5- to 2-fold (see Fig. S3 in the supplementary
material), similar to Dkk2 in Xenopus embryos
(Brott and Sokol, 2002).
The evidence for an inhibitory function in Hydra is less clear.
Nevertheless, the expression patterns of hydkk1/2/4 and
hywnt3a/hyß-catenin/hytcf are mutually exclusive and
suggestive: hywnt3a and other Wnt genes from Hydra are
expressed in the hypostomal region around the mouth of the Hydra (F.
Rentzsch, C.G., B.H. and T.W.H., unpublished)
(Hobmayer et al., 2000),
whereas hydkk1/2/4 is uniformly expressed in the entire body column,
but not in the hypostomal region (Fig.
3). This is consistent with the idea that HyDkk1/2/4 is involved
in a mechanism that might help to suppress the expression of Wnt genes in the
body column. It is also consistent with the fact that, in polyps that were
depleted of hydkk1/2/4-expressing cells, hywnt3a is
expressed in small cell clusters all over the body column. This patchy
upregulation of hywnt3a was never observed in normal polyps except in
the budding region. It should be emphasized, however, that this interaction
might be more complicated. During budding, hywnt3a-expression spots
regularly arise in a tissue that strongly expresses hydkk1/2/4
(Hobmayer et al., 2000).
hywnt3a and hydkk1/2/4 are also co-expressed during early
regeneration. Thus, although our functional assays in Xenopus provide
clear evidence that the HyDkk1/2/4 molecule has the ability to antagonize Wnt
signalling, it remains unclear as to what extent HyDkk1/2/4 actually
antagonizes Wnt signalling in Hydra.
Evolutionary considerations
Although hydkk1/2/4 is evidently required for the regeneration
process in Hydra, it remains to be clarified whether the molecule has
an additional role in steady-state animals. At this point, we can only
speculate, as no experiments addressing this question have been done. However,
the characteristic expression pattern of hydkk1/2/4 and the fact that
hydkk1/2/4 can induce the Dkk1 phenotype in Xenopus suggests
that hydkk1/2/4 has a similar function in steady-state Hydra
polyps as during head and neuronal induction in vertebrates. In vertebrates,
Dkk1 can induce secondary heads (Glinka et
al., 1997), and is necessary to activate neural genes in the
anterior and dorsal region of the vertebrate brain
(Glinka et al., 1998;
Kazanskaya et al., 2000;
Mukhopadhyay et al., 2001;
Niehrs, 2004). In Hydra,
hydkk1/2/4 is only expressed in the body column.
Figure 9 shows schematically
that in this region the neuronal differentiation from multipotent interstitial
stem cells takes place (Grens et al.,
1996; Lindgens et al.,
2004; Smith et al.,
1999; Technau et al., 1996). We therefore favour the definition of
the body column as being the neurogenic region of the Hydra. We
presume that the release of HyDkk1/2/4 from gland cells in the endoderm
promotes neuronal differentiation by inhibiting the ß-Catenin/Wnt
signalling pathway in the ectoderm of the Hydra body column
(Fig. 9A). The BMP antagonist
Chordin is also expressed in the same tissue (F. Rentzsch, C.G., B.H. and
T.W.H., unpublished), supporting the hypothesis that a primary function of Bmp
and Wnt antagonism in metazoan evolution is the facilitation of neuronal
differentiation. This hypothesis is supported by the expression patterns of
cnidarian Wnt genes (Hobmayer et al.,
2000; Kusserow et al.,
2005) and by theoretical considerations on midline formation
during the evolution of bilaterians
(Meinhardt, 2002;
Meinhardt, 2004). According to
that idea, the Hydra body column is the counterpart to the vertebrate
brain, where proneuronal and neuronal genes are expressed, whereas the
bilaterian trunk evolved later, and intercalated between the posterior and
anterior end.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/4/901/DC1
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aravind, L. and Koonin, E. V. (1998). A colipase fold in the carboxy-terminal domain of the Wnt antagonists – the Dickkopfs. Curr. Biol. 8,R477 -R478.[CrossRef][Medline]
Birnbaum, D., Coulier, F., Pebusque, M. J. and Pontarotti, P. (2000). "Paleogenomics": looking in the past to the future. J. Exp. Zool. 288, 21-22.[CrossRef][Medline]
Brott, B. K. and Sokol, S. Y. (2002).
Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins.
Mol. Cell. Biol. 22,6100
-6110.
Broun, M., Gee, L., Reinhardt, B. and Bode, H. R.
(2005). Formation of the head organizer in hydra involves the
canonical Wnt pathway. Development
132,2907
-2916.
Caldwell, G. M., Jones, C., Gensberg, K., Jan, S., Hardy, R. G.,
Byrd, P., Chughtai, S., Wallis, Y., Matthews, G. M. and Morton, D. G.
(2004). The Wnt antagonist sFRP1 in colorectal tumorigenesis.
Cancer Res. 64,883
-888.
Coulier, F., Burtey, S., Chaffanet, M., Birg, F. and Birnbaum, D. (2000a). Ancestrally-duplicated paraHOX gene clusters in humans. Int. J. Oncol. 17,439 -444.[Medline]
Coulier, F., Popovici, C., Villet, R. and Birnbaum, D. (2000b). MetaHox gene clusters. J. Exp. Zool. 288,345 -351.[CrossRef][Medline]
David, C. N. (1973). A quantitative method for maceration of Hydra tissue. Roux's Arch. Dev. Biol. 171,259 -268.[CrossRef]
Davidson, G., Mao, B., del Barco, Barrantes, I. and Niehrs, C. (2002). Kremen proteins interact with Dickkopf1 to regulate anteroposterior CNS patterning. Development 129,5587 -5596.[CrossRef][Medline]
Fedders, H., Augustin, R. and Bosch, T. C. (2004). A Dickkopf-3-related gene is expressed in differentiating nematocytes in the basal metazoan Hydra. Dev. Genes Evol. 214,72 -80.[CrossRef][Medline]
Gawantka, V., Delius, H., Hirschfeld, K., Blumenstock, C. and Niehrs, C. (1995). Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1. EMBO J. 15,6268 -6279.
Glinka, A., Wu, W., Onichtchouk, D., Blumenstock, C. and Niehrs, C. (1997). Head induction by simultaneous repression of Bmp and Wnt signalling in Xenopus. Nature 389,517 -519.[CrossRef][Medline]
Glinka, A., Wu, W., Delius, H., Monaghan, A. P., Blumenstock, C. and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391,357 -362.[CrossRef][Medline]
Gonzalez-Sancho, J. M., Aguilera, O., Garcia, J. M., Pendas-Franco, N., Pena, C., Cal, S., Garcia de Herreros, A., Bonilla, F. and Munoz, A. (2005). The Wnt antagonist DICKKOPF-1 gene is a downstream target of beta-catenin/TCF and is downregulated in human colon cancer. Oncogene 24,1098 -1103.[CrossRef][Medline]
Gregory, C. A., Singh, H., Perry, A. S. and Prockop, D. J.
(2003). The Wnt signalling inhibitor dickkopf-1 is required for
reentry into the cell cycle of human adult stem cells from bone marrow.
J. Biol. Chem. 278,28067
-28078.
Gregory, C. A., Perry, A. S., Reyes, E., Conley, A., Gunn, W. G.
and Prockop, D. J. (2005). Dkk1-derived synthetic peptides
and lithium chloride for the control and recovery of adult stem cells from
bone marrow. J. Biol. Chem.
280,2309
-2323.
Grens, A., Gee, L., Fisher, D. A. and Bode, H. R. (1996). CnNK-2, an NK-2 homeobox gene, has a role in patterning the basal end of the axis in hydra. Dev. Biol. 180,473 -488.[CrossRef][Medline]
Grotewold, L. and Ruther, U. (2002a). Bmp, Fgf and Wnt signalling in programmed cell death and chondrogenesis during vertebrate limb development: the role of Dickkopf-1. Int. J. Dev. Biol. 46,943 -947.[Medline]
Grotewold, L. and Ruther, U. (2002b). The Wnt antagonist Dickkopf-1 is regulated by Bmp signaling and c-Jun and modulates programmed cell death. EMBO J. 21,966 -975.[CrossRef][Medline]
Hansen, G. N., Williamson, M. and Grimmelikhuijzen, C. J. P. (2000). Two-color double-labeling in situ hybridization of whole-mount Hydra using RNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization. Cell Tissue Res. 301,245 -253.[CrossRef][Medline]
Hashimoto, H., Itoh, M., Yamanaka, Y., Yamashita, S., Shimizu, T., Solnica-Krezel, L., Hibi, M. and Hirano, T. (2000). Zebrafish Dkk1 functions in forebrain specification and axial mesendoderm formation. Dev. Biol. 217,138 -152.[CrossRef][Medline]
Hobmayer, B., Rentzsch, F., Kuhn, K., Happel, C. M., von Laue, C. C., Snyder, P., Rothbacher, U. and Holstein, T. W. (2000). WNT signalling molecules act in axis formation in the diploblastic metazoan Hydra. Nature 407,186 -189.[CrossRef][Medline]
Hobmayer, E., Holstein, T. W. and David, C. N.
(1990a). Tentacle morphogenesis in hydra I: The role of head
activator. Development
109,887
-895.
Hobmayer, E., Holstein, T. W. and David, C. N.
(1990b). Tentacle morphogenesis in hydra II: Formation of a
complex between a sensory nerve cell and a battery cell.
Development 109,897
-904.
Holstein, T. W., Hobmayer, E. and David, C. N. (1991). Pattern of epithelial cell cycling in hydra. Dev. Biol. 148,602 -611.[CrossRef][Medline]
Holstein, T. W., Hobmayer, E. and Technau, U. (2003). Cnidarians: an evolutionarily conserved model system for regeneration? Dev. Dyn. 226,257 -267.[CrossRef][Medline]
Horwitz, E. M. (2004). Dkk1-mediated expansion of adult stem cells. Trends Biotechnol. 22,386 -388.[CrossRef][Medline]
Jacobs, K. A., Collins-Racie, L. A., Colbert, M., Duckett, M., Golden-Fleet, M., Kelleher, K., Kriz, R., LaVallie, E. R., Merberg, D., Spaulding, V., et al. (1997). A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198,289 -296.[CrossRef][Medline]
Jacobs, K. A., Collins-Racie, L. A., Colbert, M., Duckett, M., Evans, C., Golden-Fleet, M., Kelleher, K., Kriz, R., La Vallie, E. R., Merberg, D. et al. (1999). A genetic selection for isolating cDNA clones that encode signal peptides. Methods Enzymol. 303,468 -479.[Medline]
Kazanskaya, O., Glinka, A. and Niehrs, C. (2000). The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning. Development 127,4981 -4992.[Abstract]
Klein, B., Le Moullac, G., Sellos, D. and Van Wormhoudt, A. (1996). Molecular cloning and sequencing of trypsin cDNAs from Penaeus vannamei (Crustacea, Decapoda): use in assessing gene expression during the moult cycle. Int. J. Biochem. Cell Biol. 28,551 -563.[CrossRef][Medline]
Knockaert, M., Wieking, K., Schmitt, S., Leost, M., Grant, K.
M., Mottram, J. C., Kunick, C. and Meijer, L. (2002).
Intracellular Targets of Paullones. Identification following affinity
purification on immobilized inhibitor. J. Biol. Chem.
277,25493
-25501.
Korinek, V., Barker, N., Morin, P. J., van Wichen, D., de Weger,
R., Kinzler, K. W., Vogelstein, B. and Clevers, H. (1997).
Constitutive transcriptional activation by a beta-catenin-Tcf complex in
APC–/– colon carcinoma. Science
275,1784
-1787.
Krupnik, V. E., Sharp, J. D., Jiang, C., Robison, K., Chickering, T. W., Amaravadi, L., Brown, D. E., Guyot, D., Mays, G., Leiby, K. et al. (1999). Functional and structural diversity of the human Dickkopf gene family. Gene 238,301 -313.[CrossRef][Medline]
Kusserow, A., Pang, K., Sturm, C., Hrouda, M., Lentfer, J., Schmidt, H. A., Technau, U., von Haeseler, A., Hobmayer, B., Martindale, M. Q. et al. (2005). Unexpected complexity of the Wnt gene family in a sea anemone. Nature 433,156 -160.[CrossRef][Medline]
Leost, M., Schultz, C., Link, A., Wu, Y. Z., Biernat, J., Mandelkow, E. M., Bibb, J. A., Snyder, G. L., Greengard, P., Zaharevitz, D. W. et al. (2000). Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25. Eur. J. Biochem. 267,5983 -5994.[Medline]
Leveugle, M., Prat, K., Popovici, C., Birnbaum, D. and Coulier, F. (2004). Phylogenetic analysis of Ciona intestinalis gene superfamilies supports the hypothesis of successive gene expansions. J. Mol. Evol. 58,168 -181.[CrossRef][Medline]
Li, L., Mao, J., Sun, L., Liu, W. and Wu, D.
(2002). Second cysteine-rich domain of Dickkopf-2 activates
canonical Wnt signaling pathway via LRP-6 independently of dishevelled.
J. Biol. Chem 277,5977
-5981.
Lindgens, D., Holstein, T. W. and Technau, U.
(2004). Hyzic, the Hydra homolog of the zic/odd-paired
gene, is involved in the early specification of the sensory nematocytes.
Development 131,191
-201.
