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First published online 1 February 2006
doi: 10.1242/dev.02265

Development 133, 901-911 (2006)
Published by The Company of Biologists 2006
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An ancient Wnt-Dickkopf antagonism in Hydra

Corina Guder1,2, Sonia Pinho3, Tanju G. Nacak2,4, Heiko A. Schmidt5,6,7,8, Bert Hobmayer9, Christof Niehrs3 and Thomas W. Holstein1,2,*

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.


Figure 1
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  		Fig. 1. Isolation of secreted molecules from the Hydra head organizer in a yeast signal peptide secretion screen. (A) Polyps of the temperature-sensitive strain Hydra magnipapillata sf-1 were exposed to heat shock for 3 days, causing the elimination of interstitial cells. Heads, as well as regenerating tips, were isolated from the heat-shocked polyps at various times after head removal for mRNA and cDNA synthesis. (B) Cloning strategy. Size-fractionated cDNA was cloned adjacent to a signal peptide-deficient yeast invertase gene for expression in the yeast strain YTK12. Clones expressing a fusion protein with an intact Hydra signal peptide were selected by growth on raffinose plates.

 

Figure 2
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  		Fig. 2. Sequence analysis of hydkk1/2/4. (A) TCoffee alignment of HyDkk1/2/4 with the CRD2 of available Dkk molecules (see Materials and methods). (B) Domain structure of mouse Dkk4, NvDkk1/2/4 and HyDkk1/2/4; shaded boxes indicate conserved residues within CRD2; cysteines in red. (C) IQPNNI ML tree of Dkk CRD2 domains with TREE-PUZZLE support values (>50).

 

Figure 3
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  		Fig. 3. hydkk1/2/4-expression analysis by whole-mount in situ hybridization. (A) Whole animal. (B) Double ISH: hywnt3a (blue) and hydkk1/2/4 (red). (C,D) Residual gland cells in epithelial animals of strain sf-1 after heat shock; individual gland cells are shown in D. (E-H) Animals undergoing gametogenesis. (E-G) Oogenesis; (H) spermatogenesis.

 

Figure 4
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  		Fig. 4. hydkk1/2/4 expression during budding and regeneration. (A) Early to late developing bud stages; (B) head regeneration. Polyps were decapitated at 80% body length and allowed to regenerate for the times indicated; arrows indicate the cutting position to isolate regenerating tips for quantification (see text). (C) Quantification of hydkk1/2/4-expression dynamics in regenerating tips. Expression in the apical 10% of body length was determined (n=14 to 24 polyps per sample): dark bars represent animals with hydkk1/2/4 upregulation, light bars represent silenced hydkk1/2/4 expression. (D,E) hydkk1/2/4 expression in macerated cells. bc, cells of normal body column tissue; reg, cells from regenerating tissue.

 

Figure 5
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  		Fig. 5. hydkk1/2/4-expression dynamics. (A,B) hydkk1/2/4-expression dynamics in injured (A) and ligated (B) animals after 6 hours and 1 hour, respectively.

 

Figure 6
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  		Fig. 6. hydkk1/2/4 and hywnt3a expression in regenerating epithelial sf-1 polyps. (A) Quantification of residual hydkk1/2/4-positive gland cells in body column pieces (left); n=6, 3, 5, 48, 37 and 22 with increasing time. Efficiency of head regeneration was measured as the average number of tentacles per regenerate (right); n=20, 101, 69, 178 and 140. Bars indicate s.d. Animals were cut at 50% body length and regenerated at least 4 hours prior to in situ hybridization and up to 9 days for determination of regeneration behaviour. (B-G) hydkk1/2/4 expression in individual regenerates, 4 hours (B-D) and 9 days (E-G) after head removal. (H-J) Ectopic hywnt3a expression in regenerating epithelial sf-1 polyps (5-9 days regeneration).

 

Figure 7
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  		Fig. 7. hydkk1/2/4 expression in alsterpaullone (AP)-treated animals. Polyps were incubated for 24 hours in 5 µM AP and then transferred to Hydra medium for time indicated. (A) Dark-field micrographs. (B) Quantification of hydkk1/2/4 expression (white squares) and tentacle formation (black circles) from three independent experiments, determined as the percentage of hydkk1/2/4-expressing polyps and the number of tentacles from a total of 120 polyps. Bars indicate s.d.; solid line indicates the length of AP treatment. (C) ISH with an antisense hydkk1/2/4 probe (for details see Results).

 

Figure 8
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  		Fig. 8. Heterologous expression of hydkk1/2/4 in Xenopus laevis embryos. (A) Overexpression of xdkk1 or hydkk1/2/4 induces the Dickkopf phenotype. (B) Inhibition of xwnt8-mediated secondary axis induction by xdkk1 or by hydkk1/2/4 co-injection. (C) Animal cap assay. Inhibition of siamois induction by co-injection of xdkk1 or hydkk1/2/4 was assayed by RT-PCR. WE, whole embryos; xbra, Xenopus brachyury; h4, histone-4. (D) Co-injection of hydkk1/2/4 (750 pg and 3 ng) or xdkk1 blocks xwnt8-induced activation of a siamois-luciferase reporter construct (stage 10 to 10.5).

 

Figure 9
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  		Fig. 9. hydkk1/2/4-expression pattern and neuronal differentiation in Hydra. (A) The release of HyDkk1/2/4 from gland cells in the endoderm is postulated to facilitate stem cell growth and the formation of neuronal precursor cells in the ectoderm. nvp, nerve cell precursors; nm, mature nematocytes; nmb, nematoblast; nmp, nematocyte precursors; nv, neuronal cells. (B) The hydkk1/2/4-expression domain correlates to that of neuronal and pro-neuronal genes in Hydra. Modified, with permission, from Meinhardt (Meinhardt, 2002Go; Meinhardt, 2004Go).

 




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